CN101522909A

CN101522909A - Thermal cycling system

Info

Publication number: CN101522909A
Application number: CNA2007800175485A
Authority: CN
Inventors: G·马尔特佐斯; M·约翰斯顿; D·古德温; A·谢勒; C·I·沃克
Original assignee: California Institute of Technology CalTech
Current assignee: California Institute of Technology CalTech
Priority date: 2006-05-17
Filing date: 2007-05-17
Publication date: 2009-09-02

Abstract

This invention provides a system for performing PCR, and real time PCR in particular, with great speed and specificity. The system employs a heat block containing a liquid composition to rapidly transfer heat to and from reaction vessels. The system makes use of the reflective properties of the liquid metal to reflect signal from the PCR into the vessel and out the top. In this way, the signal canbe measured by an optical assembly in real time without removing the vessels from the heat block.

Description

Heat circulating system

Cross reference

No. 60/873084 and No. 60/873172 U.S. Provisional Application No. of submitting on December 6th, 06 submitting on December 6th, No. 60/832492 1 of submitting on July 22nd, No. 60/801178 1 that the application requires on May 17th, 06 to submit to, these applications are incorporated herein by reference.

Background technology

It is one of most important scientific development of 20th century that the PCR of nineteen eighty-three Kai Limulisi (Kary Mullis) invention is described as.Round pcr is differentiated by expansion greatly and regeneration makes molecular biology that revolutionary variation take place as the ability of the genetic stocks of DNA.Nowadays, PCR has been used to carry out various tasks routinely in medical science and biological research laboratories, calculates as the detection of heredopathia, the evaluation of genetic fingerprint, the diagnosis of communicable disease, clone, paternity test and the DNA of gene.This method is by using heat-stable DNA polymerase and the machine that is commonly referred to " thermal cycler " to realize automatization.

Conventional thermal cycler exists several inherent limitations.Usually conventional thermal cycler comprises the METAL HEATING PROCESS piece to carry out the thermal cycling of response sample.Because it is relatively poor that thermal cycler has the thermal conductivity of big thermal mass and sample cell (sample vessel), it is lower therefore to carry out cycle efficiency in temperature required level.The alternating temperature speed of conventional thermal cycler is fast inadequately usually, thereby causes undesirable non-specific amplification of target sequence inevitably.Conventional thermal cycler can not reach optimum performance also owing to lack thermal uniformity, and this receives wide acceptance in this area.In addition, big and expensive optical detection elements is carried by conventional real-time thermal cycler system.In No. 6533255 United States Patent (USP)s, people such as Mitsuhashi disclose a kind of liquid metal PCR thermal cycler.

Therefore, still press for the design of alternate thermal cycler.The ideal device allows (a) to transmit heat fast and equably to produce the more specific amplified reaction of nucleic acid; And/or (b) monitor the process of amplified reaction in real time.The present invention has satisfied these and has required and provide simultaneously relevant advantage.

Summary of the invention

On the one hand, the invention provides the method for a kind of PCR of carrying out, comprise: a) in sample cell, carry out PCR, and in sample cell, produce the signal of indication PCR process, wherein all described signal reflexs are returned in the described sample cell basically, and wherein all described signals are detected from a discrete positions of described sample cell basically; And b) measures from the signal of this discrete positions emission of sample cell.In one embodiment, this method comprises a plurality of PCR round-robin of real-time measurement signal.In another embodiment, sample cell comprises the transparent wall of signal, and at least a portion of this wall immersion liquid composition, all arrive the signal of this liquid composition basically in this liquid composition reflection.In another embodiment, described liquid composition comprises metal or metal alloy.In another embodiment, signal is produced by marker or dyestuff.In another embodiment, described sample cell comprises the material of all the described signals basically in the described sample of reflection.In another embodiment, described sample cell is made of metal.In another embodiment, described sample cell comprises reflecting material.In another embodiment, described sample cell is isolated by container holder (receptacle) and described liquid composition.

On the other hand, the invention provides the method for a kind of PCR of carrying out, comprising: a) under the following conditions the target nucleotide sequences in the reaction mixture is carried out PCR reaction: i) between primer extension and double-stranded the dissociating and the rate temperature change between primer annealing and the primer extension surpass 10.5 ℃ of p.s.s; Ii) described PCR carries out in comprising the heat block of sample cell, and the temperature contrast in the sample cell is less than 0.5 ℃; And b) monitors the amplification of target nucleotide sequences in real time.In one embodiment, described heat block comprises liquid composition.In another embodiment, described method of carrying out PCR further is included in the temperature contrast measured between two or more slotted eyes of described heat block less than 0.5 ℃.In another embodiment, the described temperature contrast of measuring between two or more slotted eyes of described heat block is 0.01 ℃.In another embodiment, described heat block is a swap block.In another embodiment, variation of temperature speed realizes in the following manner: the liquid composition of the heat transfer coefficient of 0.1W/MK carries out thermo-contact to the sample cell that (1) will comprise reaction mixture with having at least; Wherein the temperature of liquid composition is controlled the temperature of reaction mixture.In another embodiment, by luring that the liquid composition motion keeps temperature homogeneity into.

On the other hand, the invention provides the method for a kind of PCR of carrying out, comprising: in thermal cycler, carry out the PCR reaction with the speed setting sample temperature that is higher than 5 ℃ of per seconds; The temperature of wherein said thermal cycler is regulated by the liquid composition in the heat block of liquid-tight seal; The heat transfer coefficient of wherein said liquid composition is at least 0.1W/MK.In one embodiment, described thermal cycler provides the alternating temperature speed at least about 10 ℃ of per seconds.In another embodiment, described thermal cycler provides at least about between 0.01 ℃ to 0.1 ℃ slotted eye and temperature homogeneity sample.In another embodiment, described heat block has 0.01 ℃ the temperature contrast of measuring between two or more slotted eyes of described heat block.In another embodiment, described liquid composition is contained in the described heat block.In another embodiment, described heat block is a swap block.

On the other hand, the invention provides the method for a kind of PCR of carrying out, comprising: regulating sample temperature fully so that carry out PCR to be at least in the thermal cycler that 3 signal index detects in real time to amplification; Wherein said detection realizes that by non-specific nucleic acid markers wherein said thermal cycler is with the speed setting sample temperature of p.s. more than 10 ℃.

On the other hand, the invention provides a kind of method of carrying out PCR in real time, comprise: a) in reaction mixture between primer extension and double-stranded the dissociating and under the condition of the rate temperature change between primer annealing and the primer extension for 5 ℃ of per seconds at least, in reaction mixture, carry out the PCR reaction of target nucleotide sequences, wherein said PCR be reflected at the situation with liquid composition generation thermo-contact of the heat transfer coefficient of 0.1W/mK at least under carry out; And b) PCR is monitored in real time.In one embodiment, described monitoring comprises: a) carry out the PCR reaction in sample cell, and produce the signal of indication PCR process in sample cell, wherein said signal is launched from sample cell from a discrete positions basically; And b) measures from the signal of this discrete positions emission of sample cell.In another embodiment, the described temperature rise rate in the reaction mixture is 40 ℃ of per seconds at least.In another embodiment, described temperature variation is by amber ear card (Peltier) element regulation.

On the other hand, the invention provides a kind of method of carrying out PCR in real time, comprising: the temperature of PCR reaction mixture is dissociated in two strands, carry out a plurality of circulations between the temperature of primer annealing and primer extension, wherein each circulation was no more than for 5 seconds; Wherein said temperature is regulated by the liquid composition that is sealed in the thermal cycler; And b) the PCR process in the real-time monitoring sample cell in a plurality of circulations.In one embodiment, the method of claim 28 further comprises before in step (a): 1) make the blind end of the sample cell that comprises the PCR reaction mixture and liquid composition form thermo-contact, this liquid composition is being liquid more than 60 ℃ and is having the heat transfer coefficient that is at least 0.1W/mK, thus the temperature of the temperature of liquid composition control PCR reaction mixture.In another embodiment, all basically light in the described liquid composition reflection sample cell.In another embodiment, described monitoring is to be undertaken by measuring from the signal of sample cell top or bottom emission.In another embodiment, each circulation is no more than 3 seconds.In another embodiment, described sample cell comprises reflecting surface.In another embodiment, described sample cell further covers with lid.In another embodiment, described lid can cool off from described liquid composition heat absorption or by described liquid composition.

On the other hand, the invention provides a kind of method of carrying out PCR in real time, comprise the thermal cycler that comprises with the liquid composition of PCR sample cell generation thermo-contact is provided; Wherein said liquid composition carries out temperature regulation, thus the temperature of reaction in the regulation and control sample cell, the wherein detectable signal of emission from described sample cell; And detect described signal by the optical module that comprises optical transmitting set and fluorescence detector.In one embodiment, described optical module comprises pin photodiode CCD imager, cmos imaging instrument, line scanner, photorectifier, phototransistor, photomultiplier cell or avalanche photodide.In another embodiment, described sample cell further covers with lid.In another embodiment, described lid can cool off from described liquid composition heat absorption or by described liquid composition.

On the other hand, the invention provides a kind of instrument, this instrument comprises: a) temperature-controlling module, comprise the container that comprises liquid composition, described liquid composition more than 60 ℃ is being liquid and the heat transfer coefficient with at least 0.1 watts/meter-Ke Shi degree (W/mK), described container has at least one hole, each hole is suitable for holding the blind end of sample cell, the sample cell and the liquid composition that wherein are contained in the hole form thermo-contact, thereby the temperature of liquid sample in the temperature of the liquid composition control sample cell, and wherein liquid composition can reflect light all basically in the sample cell; B) optical module can detect the light in the described sample cell from a discrete positions on the described sample cell; And c) control unit, the temperature of its controlled liq composition and the operation of optical module.In one embodiment, liquid composition comprises gallium, gallium indium alloy or comprises gallium, indium, rhodium, silver, zinc, tin or stannous alloy.In another embodiment, described temperature regulator comprises Peltier's element.In another embodiment, described temperature regulator comprises the resistance wire with the liquid composition thermo-contact.In another embodiment, described temperature regulator comprise that the temperature that makes liquid composition is dissociated in the two strands that is suitable for PCR, round-robin device between the temperature of primer annealing and primer extension.In another embodiment, described thermal cycler also comprises the device that the liquid stream that makes in the liquid composition circulates.In another embodiment, described optical module comprises optical transmitting set and fluorescence detector.In another embodiment, this instrument also comprises the specimen preparation station that comprises the device of interpolation reagent in sample cell.In another embodiment, described instrument further comprises sample cell is moved to device in the hole.In another embodiment, this instrument also comprises the digital machine of controlling thermal cycler, optical module and specimen preparation station.

On the other hand, the invention provides a kind of system that carries out PCR in real time, comprising: a) thermal cycler comprises liquid composition and is used to engage sample cell and makes described sample cell and composition forms the device of thermo-contact; And b) optical module comprises the sample cell and optical transmitting set and the fluorescence detector of detection from the light of the sample cell emission of joint that import light into joint.In one embodiment, described liquid composition comprises metal or metal alloy.In another embodiment, described liquid composition comprises gallium.In another embodiment, the signal of described emission is from the removable lid of described sample cell.In another embodiment, described liquid composition is isolated by container holder and described sample cell.In another embodiment, described container holder is transparent or semitransparent.In another embodiment, described system comprises Peltier's element.In another embodiment, described thermal cycler further comprises the electric motor that is operatively connected with fan and stirring rod.In another embodiment, described fan and stirring rod are connected with described electric motor is coaxial.In another embodiment, described thermal cycler further comprises the resistance wire of the heat regulation and control that are used for described liquid composition.In another embodiment, described liquid composition is sealed in the barrier of sealing, and wherein said barrier comprises the surface with container holder, and described sample cell places this container holder.In another embodiment, described liquid composition directly contacts with described sample cell.In another embodiment, described optical module comprises pin photodiode, CCD imager, cmos imaging instrument, line scanner, photorectifier, phototransistor, photomultiplier cell or avalanche photodide.

On the other hand, the invention provides a kind of instrument, this instrument comprises a) scatterer; B) heating unit, this heating unit contacts with described radiator heat; C) barrier comprises the wall with end face and bottom surface, and wherein the bottom surface is sealed on the described heating unit, and described thus sealing barrier and described heating unit form the container that comprises liquid composition; D) first lamellar body comprises a plurality of slotted eyes, and wherein this first lamellar body is sealed on the end face of barrier, and slotted eye extends in the described container; E) second lamellar body comprises a plurality of sample cells, and sample cell respectively has opening end, and wherein sample cell inserts in the slotted eye removedly; F) the 3rd lamellar body comprises a plurality of projections, and wherein projection is inserted the opening end of sample cell removedly.In one embodiment, described liquid composition is included in more than 60 ℃ to liquid and has the metal of the heat transfer coefficient that is at least 0.1M/W/K.In another embodiment, the volume of described liquid metal can increase about 0.1 to 6.0%.In another embodiment, described intensification ability surpasses 10.5 ℃ of per seconds.In another embodiment, e) described in slotted eye be set to Yi Shixing (flexible), thereby described slotted eye can with f) described in sample cell form heat or optics contacts.In another embodiment, described setting is included in and utilizes geometric configuration and/or deformable material in the described slotted eye.In another embodiment, described geometric configuration is Polygons, ellipse or circular.In another embodiment, described slotted eye immerses described liquid composition and reaches initial level.In another embodiment, described projection extend to that described initial level is above, described initial level place or below the described initial level.In another embodiment, described the 3rd lamellar body can cool off from described liquid composition absorption heat or by described liquid composition.In another embodiment, the slotted eye of described first lamellar body is transparent and Yi Shixing (flexibility), and the sample cell of described second lamellar body is transparent, makes described liquid and sample in the described sample cell in the described container form light and heat to learn and contact thereby act on enhanced pressure on the described slotted eye.In another embodiment, the projection of described the 3rd lamellar body is transparent.In another embodiment, described barrier forms sealing with the radiator and first lamellar body respectively by pad.In another embodiment, described first lamellar body or second lamellar body have reflecting surface.In another embodiment, described barrier forms airtight with the radiator and first lamellar body respectively by fastener.In another embodiment, described instrument also comprises the radiator that contacts with described heating unit.In another embodiment, described heating unit is Peltier's element, kind of thread elements or its combination.In another embodiment, described instrument further comprises second heating unit, and described barrier is between described heating unit and described second heating unit.In another embodiment, described a plurality of slotted eye comprises 4,8,16,32,48,96,196,384 or 1536 slotted eyes.In another embodiment, described instrument further comprises fan and stirring rod, and wherein randomly described fan and stirring rod and monomotor are operatively connected.In another embodiment, described instrument provides power by battery.In another embodiment, described fan is opened and closed automatically by the controlling elements that is connected with described instrumentation.In another embodiment, described instrument also comprises the heat dissipation metal body.In another embodiment, described radiator comprises copper.In another embodiment, described disposition of heating component is used for grads PCR.

On the other hand, the invention provides Continuous Flow PCR system, it comprises: the specimen preparation module; Thermal cycler, wherein said thermal cycler comprises the liquid composition in order to the temperature of regulating described sample; And optical module, be used to detect transmitting of described sample.In one embodiment, described system also comprises sampling thief and waste collection device.

Introduce by reference

All publications mentioned in this manual and patent application are here introduced by reference, show introducing by reference especially and individually as each single publication or patent application.

Brief Description Of Drawings

New feature of the present invention is listed in the appended claims especially.In order to understand characteristics of the present invention and advantage better, please refer to following detailed description and following accompanying drawing to the illustrated embodiment that adopted principle of the present invention:

Fig. 1 illustrates the thermal cycler main body that adopts liquid composition exchange heat block.(A) described 48 holes " swap block " device on the slide rail.This piece skids off, and load sample slot orifice plate and sample cell orifice plate lid slips in the thermal cycler main body then and closes the door.The thermal cycler main body can comprise optical module, electronic regulator, fan and optional power supply.(B) " swap block " slips into the thermal cycler main body of working position.

Fig. 2 illustrates the swap block embodiment that comprises 48 sample slotted eyes.In this embodiment, this swap block comprises from top to bottom: as the monolithic body of 48 transparent cover body; Produce the monolithic body of 48 sample slotted eyes; Single container holder lamellar body with 48 reacting holes, it forms the loam cake of liquid metal vessels; Form the plastic casing of the wall of liquid metal vessels; Form the metal sheet of liquid metal vessels base plate; The amber ear card device that is used for heating and cooling; And be the metal heat sink of amber ear card device radiation.

Fig. 3 is the exploded view of swap block embodiment.In this embodiment, swap block comprises from top to bottom: as the monolithic body of 48 transparent caps; Monolithic body as 48 transparent cover body; Produce the monolithic body of 48 sample slotted eyes; Single container holder lamellar body with 48 reacting holes; Rubber or plastic packing or ring, its formation is used for holding the fluid-tight of liquid metal; Form the plastic casing of the wall of liquid metal vessels; Rubber or plastic packing or ring, its formation is used for holding the fluid-tight of liquid metal; And the metal sheet of optional formation liquid metal chamber base plate; Be used for the amber ear card device of heating and cooling; And be the metal heat sink of amber ear card device radiation.

Fig. 4 is second view of swap block embodiment.In this embodiment, swap block comprises from top to bottom: as the monolithic body of 48 transparent cover body; Produce the monolithic body of 48 sample slotted eyes; Single container holder lamellar body with 48 reacting holes; Rubber or plastic packing or ring, the fluid-tight of liquid metal is held in its formation; Form the plastic casing of the wall of liquid metal vessels; Rubber or plastic packing or ring, the fluid-tight of liquid metal is held in its formation; Form the metal sheet of the base plate of liquid metal chamber; The amber ear card device that is used for heating and cooling; And be the metal heat sink of amber ear card device radiation.

Fig. 5 is the view of the sandwich embodiment of thermal cycler parts, and thermal cycler comprises the heat block that comprises liquid metal.(A) this view demonstrates at two amber ears card devices (two big walls) and is configured between the brown plastic sheet of square plastic ring (four narrow walls) and formed the liquid metal chamber.The liquid metal chamber comprises the hole that is used for the capillary sample hose.And, each amber ear card device and radiator heat coupling, scatterer is connected with fan again.(B) be interposed in the feature of the heat block middle body between the amber ear card device.(C) the liquid metal chamber comprises the hole that is used for the capillary sample hose.Kapillary slides in the hole, and directly immerses in the liquid metal to carry out thermal cycling.Amber ear card device is placed in the both sides of liquid metal bank, to allow to carry out apace heating and cooling.

Fig. 6 illustrates detection by the embodiment that carries out the signal that the round-robin sample cell sends in the heat block that comprises the liquid metal composition.LED is used for exciting the sample that is included in the sample cell, and the signal of any generation is detected by pin photodiode.

Fig. 7 illustrates the pcr amplification that carries out with HBV virus-specific primer and patient blood sample, and wherein PCR carries out on Roche Lightcycler.The positive peak (green) of melting curve illustrates that the HBV test is positive, but negative control peak (brown) is remarkable too.

Fig. 8 illustrates the pcr amplification that carries out with same primers as of using among Fig. 7 and sample, and wherein PCR utilizes and comprises that the thermal cycler of liquid metal heat block carries out.Positive peak (green) illustrates that the HBV test is positive, and negative control peak (brown, pink) is more much lower than Fig. 7, and the detected result of this explanation HBV is more accurate.

Fig. 9 illustrates the result of the PCR that carries out in the thermal cycler that comprises the liquid metal heat block, itself and the result's contrast that is obtained by Roche Lightcycler.Set up melting curve simultaneously with two samples: one is to obtain (green) at the enterprising performing PCR of Roche Lightcycler, and one is to obtain (brown) at the enterprising performing PCR of instrument of the present invention.Roche PCR has produced extra peak under the temperature of expression formation primer dimer, the PCR that this explanation is carried out in the Roche instrument is not as comprising that the PCR that is carried out in the thermal cycler of liquid metal heat block is accurate.

Figure 10 illustrates the embodiment of the liquid composition heat block that adopts resistance wire heating liquid composition.Top view shows that resistance wire is arranged as provides even heating, wherein place with higher density near liquid composition chamber edge resistance wire, and near centre portions, resistance wire is to place than low density.This arrangement has compensated in conventional thermal cycler design because any possible fringing effect that thermosteresis causes.As shown in side-view, resistance wire is suspended in the liquid composition, rather than is rested on the bottom, so liquid metal is from all contacts side surfaces resistance wires, and this makes and reaches maximum heat passage homogeneity.

Figure 11 illustrates the purposes that the liquid composition heat block is used for continuous P CR device.A plurality of samples continuously by described device to detect one or more specific biological specimen, for example pathogenic agent or biological pollutant.

Figure 12 illustrates the melting curve that carries out the sample of pcr amplification in the stainless steel sample cell, itself and conventional glass capillary contrast.The melting curve of two reactions illustrates and increases successfully much at one.The result of the PCR reaction of (A) in conventional thermal cycler, carrying out with the metal sample groove.40 circulations are carried out in the PCR reaction, use the generation of the green detection pcr amplification of SYBR then.(B) result of the PCR reaction of in conventional thermal cycler, carrying out with the sample cell that is purchased.40 circulations are carried out in the PCR reaction, use the generation of the green detection pcr amplification of SYBR then.

Figure 13 illustrates the preparation method of metal sample groove.This method by in a step by the raw metal cutting that draws and curl the metal sample groove and fast, production sample groove at an easy rate.Groove is used for preventing that the sample cell wall from being evened up.

Embodiment

The invention provides a kind of equipment, it comprises and is used for the sample cell that comprises reaction mixture is carried out the thermal cycler of temperature cycle, the control device that is used for detecting from the optical module of the signal of sample cell and is used to control the operation of thermal cycler and optical module.In specific embodiment, thermal cycler adopts the heat block that comprises the liquid composition (for example liquid metal or heat-conducting fluid) with high thermal conductivity to circulate with the temperature that makes sample cell apace.Use liquid metal to have 2 main advantages: the first, metal has high thermal conductivity, thereby provides heat passage fast; The second, liquid makes to form between sample cell and the thermally conductive material and contacts more closely, thereby the heat passage of homogeneous more is provided.Therefore, the temperature in the sample cell is very even.The combination of alternating temperature speed and temperature homogeneity has fast reduced non-specific hybridization and has also improved in the single sample groove significantly and the specific amplification (for example signal to noise ratio) of the reaction of the PCR between a plurality of sample cells in same heat block.In another embodiment, sample cell (combining separately or with thermal cycler) is launched all signals that wherein produce basically by a discrete parts (for example, the top of sample cell) of sample cell, thereby the light that sends can be collected by optical module.In another embodiment again, fluorescence detector detects basically all from light of sample cell emission.In some embodiments, liquid metal or sample cell are highly reflectives, and will return in the sample cell through the luminous reflectance of the wall of transparent sample groove.In this way, the major part of the optical signal that produces in the sample cell is launched from a discrete parts of sample cell, thereby can be collected by optical module.The ability of collecting more light from reaction means can select more cheap optics for use in device, thereby has reduced cost.And, collect light from the discrete positions of sample cell and make and when carrying out PCR in real time, do not need to remove sample cell from heat block.Therefore, the configuration of this heat block makes it possible to obtain fast alternating temperature speed and uniform temperature, and reaching optical module can and not need sample cell is removed from heat block from sample cell top collection reflected light, thereby makes PCR in real time carry out quickly.Correspondingly, instrument of the present invention is through being provided with to carry out PCR (polymerase chain reaction), reverse transcription PCR and PCR in real time especially.The thermal cycler that comprises the liquid metal heat block will carry out the PCR reaction more fast and economically than existing instrument on the market.In one embodiment, the thermal cycler that comprises the heat block that comprises liquid composition uses battery that power is provided.In another embodiment, the thermal cycler that comprises the heat block that comprises liquid composition uses direct current or alternating-current that power is provided.

Except the PCR heat block, liquid metal heat block of the present invention can also be widely used in biotechnology and chemical field, example is hatched including, but not limited to enzyme reaction, for example restriction enzyme, biochemical analysis and polymeric enzyme reaction, cell cultures and conversion, hybridization, and any processing that needs precise dose control.Based on disclosure of the present invention, those of ordinary skill in the art can be applied to the liquid metal technology the various analyses that need the biology/chemical example of precise temp control at an easy rate.

Thermal cycler

Utilize liquid composition (for example liquid metal or heat-conducting fluid) to obtain than the more uniform heat passage and circulation of heating and cooling faster of solid metal heat block as the heating and cooling medium of heat block.Be used for the embodiment of thermal cycler at the liquid metal heat block, heat up faster and better thermal uniformity make archaeal dna polymerase produce error rate (Fig. 7-9) lower when using conventional thermal cycler.This is because the PCR sample has been in the time decreased under the non-ideal temperature.In addition, because the raising of thermal uniformity, error rate reduces in long amplification, SNP evaluation and sequencing reaction process.

In one embodiment, liquid metal or heat-conducting fluid are used as the heating and cooling medium of heat block, and wherein heat block comprises that the end and wall are to hold liquid metal or heat-conducting fluid at least.Liquid metal or heat-conducting fluid provide on whole heat block than conventional heat block more uniform temperature, and this is because the heat passage faster and ability of utilizing convection current or stirring the distribution heat in liquid metal or the heat-conducting fluid.

In another embodiment, heat block comprises liquid metal or the heat-conducting fluid that directly contacts with sample cell.In this embodiment, can realize and the comprehensive engagement of sample cell that whatsoever the sample cell of type, size, shape all can obtain the heat passage of homogeneous.Do not need the slotted eye that is shaped in advance.And, can realize the homogeneity of temperature in the heat block, because liquid metal or heat-conducting fluid be easy to by convection current or by external strength at the heat block internal recycle, these external strengths are including, but not limited to stirring rod, pump, shaking device or magnetic fluid (MHD) power.

In another embodiment again, liquid metal or heat-conducting fluid are contained in fully in the heat block and do not contact sample cell.In this embodiment, container has the container holder that is designed to hold the sample cell with desirable shape and size.These container holders are designed to closely cooperate and are placed in sample cell in the slotted eye.In one embodiment, container holder uses and makes the light that is transmitted to wherein be made by the material of the substantial transparent in the liquid metal reflected back sample cell.In the alternate embodiment, container holder is made by the reflecting material that the luminous reflectance that produces in feasible all light that enter basically or the sample cell in the container holder slotted eye returns in the described sample cell.In another embodiment, container holder is made by both light tight also unreflecting basically opaque materials.

In one embodiment, container holder is made of flexible materials, thereby the slotted eye of container holder is suitable for closely cooperating with sample cell.Along with heat block increase in temperature and liquid metal or heat-conducting fluid volumetric expansion, the compactness that contacts between container holder slotted eye and the sample cell increases.Container holder can be made by transparent, opaque, inferior transparent or semitransparent material.

In open or enclosed embodiment, heat block can randomly comprise the storage vault of liquid metal or heat-conducting fluid, it can be the part of heat block, for example at bottom or the depression on the sidewall or the salient of heat block, the independent storage vault of perhaps using junctor (such as pipe) to link to each other with heat block.This storage vault can randomly be connected on the heat block by pump.Depend on its design, storage vault can be as spillway to collect any liquid metal that overflows or heat-conducting fluid when liquid metal or heat-conducting fluid expand the capacity limit that exceeds heat block, to keep or to change the interior pressure of heat block sealing or sealing, perhaps be used as the savings storehouse of the liquid metal of loss in use.

In one embodiment, storage vault is used to replenish liquid metal or heat-conducting fluid in the operating process of heat block.The storage vault that contains extra liquid metal or heat-conducting fluid can provide additional when loss occurrence.This replenishment system can keep liquid metal in the ideal level, perhaps keeps liquid metal or heat-conducting fluid at ideal pressure in the embodiment of sealing.In one embodiment, provide and be used for the level of pyroelectric monitor liquid metal or heat-conducting fluid and cross the detection system of reporting to the police to the operator when low in this level.This replenishment system can be taked any form easily, for example is equipped with the attached storage vault of sidepiece or bottom of piston or the pump that is used for mobile liquid metal or heat-conducting fluid, the controller of operating this piston or pump and transmitter.For example, transmitter can comprise and be positioned at the electric wire of determining the height location place, its when liquid metal reaches the height that needs with liquid metal formation loop.In another embodiment, transmitter can comprise light beam, thereby if fluid level is too low, transmitter is shone and illuminated to light beam level on the top of the cavity that liquid metal is housed.In another embodiment, transmitter comprises two light beams, one on another, low light beam represents that low-level and high light beam represents high level.Signal can be transferred to operator or the control device level with indication liquid metal or heat-conducting fluid.Signal can prompting operation person should be taked corrective action (by improving or reduce the level of liquid metal or heat-conducting fluid).In another embodiment, Controlling System is regulated the level of liquid metal or heat-conducting fluid automatically to reach the ideal level.In another embodiment again, liquid metal or heat-conducting fluid heat block comprise that the user can replenish the chamber that the user of liquid metal or heat-conducting fluid can contact as required.In one embodiment, the chamber that the user can contact links to each other with liquid metal or heat-conducting fluid heat block chamber in the bottom, and can fill in from the top.

Swap block

In one embodiment, the thermal cycler main body (101; 151) comprise fan (103; 153) reach movably heat block assembly, or swap block (105; 155) (Fig. 1).Swap block (105; 155) pass through randomly at slide rail (113; 163) go up slip exchange heat block and be inserted into thermal cycler main body (103; 153) shift out in or therefrom.When swap block (105; 155) be inserted into thermal cycler main body (103; 153) after in, thermal cycler (115; 165) door can be closed.Exchange heat block (105; 155) comprise liquid combination container (111; 161) and scatterer (107; 157) and optional sample (109 of adding a cover; 159).In one embodiment, exchange heat block (Fig. 2) comprises the container holder that has the slotted eye of liquid composition sealing, thus sample cell contact liq (metal, metal alloy or metal slurry) not.In another embodiment, swap block (105; 155) comprise having to liquid composition shell of tank (311; 411) the container holder barrier (307 of Mi Feng slotted eye; 407), wherein sealing is that liquid is close and can randomly comprise packing ring (309; 409) (Fig. 3 and Fig. 4).In addition, the liquid composition shell of tank (311; 411) be sealed to base plate (313; 413) on, base plate can be metal sheet (as copper or an aluminium), and wherein sealing is that liquid is close and can randomly comprise packing ring (312; 412).Base plate (313; 413) again with Peltier's element (315; 415) thermal coupling, the heating and cooling liquid composition also is coupled with scatterer (417) again.Randomly, radiator (having the metal or metal alloy of high thermal conductivity as copper, aluminium or other) is interposed in base plate (313; 413) and Peltier's element (315; 415) between.In some embodiments, swap block (105; 155) by fastening piece such as screw (301; 401) combine.In one embodiment, swap block comprises first lamellar body, as has the container holder (307 of 48 slotted eyes; 407), (for example sample cell includes but not limited to sample panel (305 to this first lamellar body by second lamellar body; 405), monocyte sample groove or sample troughed belt) occupy, the 3rd lamellar body is (as transparent cover plate (303; 403), single lid or lid band) insert in second lamellar body.In one embodiment, transparent cover plate (303; 403), single lid or lid band randomly comprise projection, as optical conductor.

In another embodiment, sample cell directly places liquid.In another embodiment again, container holder has and plays squeegee and do in order to wipe clean the sample cell of removing and randomly play sealing process liquid metal is sealed in inner ring (for example, O shape ring) from liquid.In another embodiment, container holder provides the sleeve pipe that sample cell is placed in one (as by the film formed sleeve pipe of flexible plastics), and in other words, sleeve pipe plays the effect of the barrier between sample cell and liquid metal or the heat-conducting fluid.

Liquid composition (as liquid metal or heat-conducting fluid) heat block keeps uniform temperature on whole.In one embodiment, this is by being realized by power, such as the convection current or the passive conduction of liquid metal or heat-conducting fluid.In an alternate embodiment, by using as the method for stirring rod or utilize pump or the recycle system of MHD power active mixes liquid metal or heat-conducting fluid strengthen the homogeneity of temperature.

In one embodiment, with pump circulate liquid metal or heat-conducting fluid in the heat block.Can mobile liquid metal or the design of any pump of heat-conducting fluid all be fit to for example positive-displacement pump (including but not limited to rotary-type pump, Reciprocatory pump, roots pump, syringe pump, Wendelkolben pump or Screw disk-seal tube pump (helical twisted roots pump)), impeller pump, MHD pump or power-driven pump.In this embodiment, pump will by can tolerable temperature difference and/or the material of the etching problem relevant with liquid metal or heat-conducting fluid make.

In another embodiment, pump is used to circulating liquid and/or makes liquid metal or heat-conducting fluid (for example, increasing the pressure of storage vault chamber) oppress container holder wall or sample wall, and this depends on the structure (for example, open or closed) of thermal cycler.For example, in closed system, pressure boost has caused liquid to exert pressure to the container holder wall, therefore causes this container holder wall more closely to be pressed against on the sample cell wall, engages thereby form.This joint has strengthened thermal conductivity and/or optical transmittance.In the embodiment that uses open system (for example, sample cell directly contacts with liquid composition), pump can improve the level of liquid metal or heat-conducting fluid to increase the surface area that it contacts with the container holder wall.

In another aspect of this invention, sample is directly put into liquid (metal, metal alloy or metal slurry), and this sample cell is subjected to the buoyancy that equates with the liquid that arranges naturally.In various embodiments, clip, top cover, fastening piece, weight, Spring Card, thread plate or this type of device are used for guaranteeing the sample cell strong fix.

In an alternate embodiment, MHD power is used to the liquid metal that circulates.Liquid metal is exposed in magnetic field that forms in one direction and the electric field that forms on the direction perpendicular to field direction.Liquid metal is along the direction perpendicular to field direction and direction of an electric field is mobile simultaneously then.By applying magnetic field simultaneously and electric field, liquid metal is flowed according to specified direction and do not need physics control (for example controlling) by pump.Thereby these field characteristics can be used for introducing liquid stream at heat block keeps uniform temperature, perhaps is used for liquid metal being introduced heat block and liquid metal for example being expelled to from heat block in the storage vault.In relevant embodiment, form electric field by direct current or alternating-current, along with frequency is regulated, liquid metal vibrates with respect to frequency change, therefore reaches the high homogeneity of temperature on whole heat block.

In another embodiment, liquid metal or heat-conducting fluid can circulate by stirring rod, and this stirring rod can link to each other with electric motor, perhaps can be magnetic response and stir in response to the variation in magnetic field.In one embodiment, stirring rod is that anti-fast temperature changes, or scribbles the tectum that anti-fast temperature changes.In one embodiment, stirring rod is simple cross bar.In the alternate embodiment, stirring rod can be flaabellum shape or have a plurality of emergences (for example 3,4,5,6,7,8,9,10,11 or 12) that are used to stir liquid metal or heat-conducting fluid.In one embodiment, thermal cycler (101) comprises the electric motor that is operatively connected with fan (103) and stirring rod.In another embodiment, fan links to each other and randomly rotation simultaneously with stirring rod and same electric motor are coaxial.

In another embodiment again, liquid metal or heat-conducting fluid can circulate by vibrating device.Vibrating device can be integrated in thermal cycler or the heat block structure, perhaps can be the auxiliary equipment that contacts with heat block or thermal cycler.Vibrating device transmitting vibrations ripple helps its convection current and shortens metal to reach the even spent time of heat by liquid metal or heat-conducting fluid.In one embodiment, acoustic apparatus is used for vibrating liquid metal or heat-conducting fluid, as piezoelectricity mixing tank (piezo mixer), ultrasonic vibrator, subsonics vibrator or other acoustic apparatus.Vibrator can comprise loudspeaker coil or piezo-electric device or mechanical motor.Therefore sample and the PCR reagent of vibrating device in also can the vibrating example groove make reactant mix, thereby make reaction more effectively take place.

Liquid metal or heat-conducting fluid are flowable in the thermal cycler operational process, and have the boiling point that is higher than service temperature.In addition, liquid metal or heat-conducting fluid are nontoxic under operating environment preferably.Liquid metal has high thermal conductivity and specific conductivity, therefore may be very sensitive to heating and cooling pattern/circulating reaction.For polymerase chain reaction (PCR), high heating and cooling speed is preferred, and liquid metal or heat-conducting fluid can satisfy this point.

Sandwich heat block

In another aspect of this invention, thermal cycler comprises sandwich liquid composition heat block (Fig. 5).In one embodiment, the sandwich heat block comprises and amber ear card heating and cooling element (503; 553; 573) the liquid combination container (501 of thermal coupling; 551; 571), amber ear card device again with scatterer (505; 555; 575) thermal coupling and randomly with side fan (507; 557) coupling.In this embodiment, liquid combination container (501; 551; 571) comprise the opening (559 that is used for sample cell (as kapillary (581)); 579).In one embodiment, sample cell contacts with liquid composition indirectly.In the alternate embodiment, between sample and the liquid composition by the deformable tube physical isolation of heat conduction.In one embodiment, scatterer comprises that pipe or radiator element are to increase the radiating surface of scatterer.In another embodiment, the opening (559 that is used for sample cell; 579) comprise rubber or plastics O-type ring, it is used for forming sealing around the sample cell of putting into liquid composition by this opening, and is used for wiping when sample cell shifts out heat block the liquid composition that may stick on the sample cell.Sandwich heat block embodiment can comprise the opening that is used at least one sample cell, as is used for the opening of 2,3,4,5,6,7,8,10,12,16,20,24,30,36,42,48,54,60,66,72,78,84,90,96,192 or 384 sample cells.

In another embodiment, all form the hole in both sides so that the top of sample cell and bottom all are visible.In such embodiment, liquid is wrapped in the plastics barrier with the hole that penetrates (for example multiple-grooved hole configuration) so that sample cell is placed in the slotted eye.In this configuration, photorectifier and fluorescence detector can be arranged on the relative both sides of given slotted eye.This makes it possible to by inducing optical signal to enter the part (for example top) of sample cell and detecting any signal that sends (vice versa) from the opposite side of piece (for example bottom) and carry out in real time or quantitative PCR.

In one embodiment, the kapillary of one or more thermal conductivity plastics (581) is for example by Cool Polymers company (333 Strawberry Field Rd., Warwick, RI02886 USA; Http:// www.coolpolymers.com) kapillary that provides is inserted into and comprises liquid metal (501; 551; 571) in the heat block.In one embodiment, kapillary (581) can see through the opposite side of described heat block, and this makes the top of pipe and bottom all is that optics can reach.In this embodiment, the sample in the pipe (581) can at one end be excited, and consequent any signal can detect at the other end.In one embodiment, pipe (581) directly contacts liquid metal.In the alternate embodiment, pipe (581) is inserted in the high thermal conductivity deformable tube (for example plastics tubing) that is included in the interior liquid metal of heat block.In this embodiment, it is possible being inserted into sample cell in the high thermal conductivity deformable plastic pipe.Subsequently, liquid metal can adopt the pump pressurization, and wherein pressure extrusion thermal conductivity plastics tubing is so that it forms tight the contact with sample cell.

The Continuous Flow heat block

In the alternate embodiment, liquid metal or heat-conducting fluid heat block can be used for continuous P CR thermal cycler.Continuous P CR thermal cycler can be high-sensitive in hope or the situation of high-throughout PCR in use.Under many situations, people may want in sensitive pcr analysis air, blood, water or other medium to be sampled continuously.This can be used to seek the various biological pollutants that comprise influenza virus, bacterial pathogens and any amount of virus or bacterial pathogens.Continuous P CR makes PCR to implement in automatic mode and does not need manual intervention.The PCR system of continuous sampling can also be used as the very early warning system in the HVAC system of buildings, aircraft, motorbus and other launch vehicle, and can be used for monitoring of blood, water and other possible source of pollution.

In one embodiment, continuous P CR system obtains sample (1101) (Figure 11) from collection device (as air sampler, fluid sampler or other sampling thief).In other embodiments, the condensed fluid of collecting on the air-conditioning system condensing unit is used as initial sample, or uses the special gas sampling assembly by direct impact work to obtain sample.Sample is produced (1103), and specimen preparation may comprise lysis, DNA or RNA purifying, filtration and/or reverse transcription in some embodiments.Prepare to be used for PCR (1105) by sample being joined in PCR reagent (for example at least a archaeal dna polymerase, dNTPs, damping fluid and salt) and the primer (for example analyze Auele Specific Primer or be widely used in the primer sets of plurality of target pathogenic agent) afterwards.These primers can be selected optionally to increase from special pathogen (as mould, virus, bacterium, parasite or amoeba) separated DNA or cDNA, gene, other required nucleic acid or its arbitrary combination.

PCR sample/reagent mixed liquor (1107) flow to thermal cycling unit (1109) by the road then.In some embodiments, described pipe is limpid or transparent.In another embodiment, pipe is opaque.In one embodiment, pipe is a right cylinder.In another embodiment, tube section comprises the one or more planes that form as trilateral, square, rectangle, pentagon, hexagon, heptagon, octagon, nonagon, decagon or other polygonal shape.In one embodiment, the volume of sample (1107) makes it occupy the space of a bit of discrete length in the sample cell, and other parts occupy by air, gas or as the non-reaction liquid of mineral oil.Pressurized gas or liquid are used for sample is pushed the heat block of thermal cycler.In one embodiment, heat block is liquid metal or heat-conducting fluid heat block, and it can be included but not limited to the Peltier's heat electricity module of thermal coupling, conventional electrothermal module, warm air or incandescence by various device heating and cooling.In one embodiment, thermal cycler adopts the outside Peltier's heat electricity module of pipe heating and cooling sample on demand.

After needed times of thermal cycle was finished, sample is compressed air in pipe or liquid further advances, thereby withdrawed from thermal cycling zone and enter detection zone (1113), can carry out fluorescence measurement, absorbance measuring here or other detects measurement.In one embodiment, except that detection zone be limpid or substantial transparent, pipe is opaque.In detection zone, light source is (as coherent source, include but not limited to laser) be used for exciting the fluorescence dye of PCR sample (as intercalative dye, include but not limited to ethidium bromide or Syber green dye and related dye), and exciting light is detected by photoelectric detector (as CCD, CMOS or other fluorescence detector).The definite signal that sends from surveyed area (1113) of detection. electronics (1115).Male PCR test will produce more substantial detection fluorescence than the PCR test of feminine gender.

Next, sample further advances in pipe, is finally collected by waste collection device (1117) as refuse.In one embodiment, pipe only is used for single to be used, and abandons then.In the alternate embodiment, whether pipe can be used for increasing in a plurality of samples and detect amplified production exists.Sample is loaded at certain intervals, and separates to prevent mutual mixing with the barrier of gas or liquid.Sample separates in transfer line, thereby makes that each sample can be by cyclic amplification and detection individually.In one embodiment, sample separates in the following manner: when a sample carried out thermal cycling, another sample was just being accepted detection in detection zone.In another embodiment, a plurality of pipes can parallel use to increase the sample flux.In another embodiment again, when thereby amplification (positive findings) took place shows that target sequence exists, the user can warn in system.

The gradient-heated piece

In the alternate embodiment, liquid metal or heat-conducting fluid heat block are designed to and can keep different temperature in the different zones of heat block, these feasible different sample cells that are arranged in the slotted eye of different zones can circulate simultaneously with different temperature, for example in the grads PCR process.In one embodiment, liquid metal or heat-conducting fluid heat block can be 2 or more interregional maintenance thermogrades, as 3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23 or 24 zones.In one embodiment, heat block comprises the container holder that has one or more sample slotted eye in each temperature province, as 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23 or 24 slotted eyes.In one embodiment, on liquid metal or heat-conducting fluid heat block, can realize surpassing 0.1 ℃-20 ℃ thermograde.

In one embodiment, thermograde upward changes with linear mode in the single dimension (level or vertical) of liquid metal or heat-conducting fluid heat block.For example comprising container holder (307 with 48 slotted eyes; 407) in the heat block, 8 rows of equidistant intervals are arranged, every row 6 holes on piece.If the top, horizontal ground at heat block forms 8 ℃ thermograde, each stock layout product slotted eye will have general 1 ℃ temperature contrast.

In some embodiments, heat block will comprise internal partition or thermal wall, and it is used for the liquid metal of different zones or heat-conducting fluid and other zone isolation.Each zone can comprise further that independent hot mixing tank (for example pump, stirring rod or MHD) or whole heat block can be connected on the single hot mixing tank (as MHD or vibrating device).In addition, each zone of heat block can comprise independent heating and/or cooling element, for example heat conducting element (line, pipe), feed thin foil strips type well heater, Peltier's element or cooling unit.

In the alternate embodiment, heat block can comprise a plurality of heating and cooling devices, and some of them are brought into play function all sidedly at all liquid metals or heat-conducting fluid heating zone, and some of them only act in the single area.For example, heat block can adopt even heating on the whole length of piece and width, and making extra heating and/or refrigerating unit each zone in abutting connection with heat block, such arrangement can be carried out more accurate adjusting to the temperature of reaction of the temperature of liquid metal in each zone or heat-conducting fluid and the associated sample groove in described zone.

Thermal conductivity

Compare with the solid metal heat block, the liquid composition heat block that comprises liquid metal or heat-conducting fluid keeps the more temperature of homogeneous on whole.The solid heat block on whole and shown aspect the sample room difference two significant temperature contrast (Schoder etc., J.Clin.MicroBiol.2005,43,2724-2728).The temperature contrast of any set point is all greater than liquid metal or heat-conducting fluid heat block in the heating installation of conventional solid metal thermal cycling instrument.Conventional thermal cycler is observed up to+/-3.3 ℃ temperature contrast, yet the maximum difference of liquid metal or heat-conducting fluid heat block temperature is also much lower than this.In one embodiment, heat block comprises the liquid metal that has greater than the heat transfer coefficient of 0.1 watts/meter-Ke Shi degree (W/mK), between 0.25-85W/MK, include but not limited to 0.25W/MK, 0.5W/MK, 0.75W/MK, 1W/MK, 1.5W/MK, 2W/MK, 2.5W/MK, 3W/MK, 3.5W/MK, 4W/MK, 4.5W/MK, 5W/MK, 5.5W/MK, 6W/MK, 6.5W/MK, 7W/MK, 7.5W/MK, 8W/MK, 8.5W/MK, 9W/MK, 9.5W/MK, 10W/MK, 11W/MK, 12W/MK, 13W/MK, 14W/MK, 15W/MK, 16W/MK, 17W/MK, 18W/MK, 19W/MK, 20W/MK, 21W/MK, 22W/MK, 23W/MK, 24W/MK, 25W/MK, 26W/MK, 27W/MK, 28W/MK, 29W/MK, 30W/MK, 32W/MK, 35W/MK, 37W/MK, 40W/MK, 42W/MK, 45W/MK, 47W/MK, 50W/MK, 55W/MK, 60W/MK, 65W/MK, 70W/MK, 75W/MK, 80W/MK, 85W/MK.

In one embodiment, liquid metal or heat-conducting fluid heat block are the parts of thermal cycler, it has sufficient thermal uniformity on whole heat block, wherein the temperature between any two sample cells has and is no more than+/-0.6 ℃ difference in the heat block, for example is no more than+/-0.59 ℃, 0.58 ℃ of+/-, 0.57 ℃ of+/-, 0.56 ℃ of+/-, 0.55 ℃ of+/-, 0.54 ℃ of+/-, 0.53 ℃ of+/-, 0.52 ℃ of+/-, 0.51 ℃ of+/-, 0.5 ℃ of+/-, 0.49 ℃ of+/-, 0.48 ℃ of+/-, 0.47 ℃ of+/-, 0.46 ℃ of+/-, 0.45 ℃ of+/-, 0.44 ℃ of+/-, 0.43 ℃ of+/-, 0.42 ℃ of+/-, 0.41 ℃ of+/-, 0.4 ℃ of+/-, 0.39 ℃ of+/-, 0.38 ℃ of+/-, 0.37 ℃ of+/-, 0.36 ℃ of+/-, 0.35 ℃ of+/-, 0.34 ℃ of+/-, 0.32 ℃ of+/-, 0.31 ℃ of+/-, 0.3 ℃ of+/-, 0.29 ℃ of+/-, 0.28 ℃ of+/-, 0.27 ℃ of+/-, 0.26 ℃ of+/-, 0.25 ℃ of+/-, 0.24 ℃ of+/-, 0.23 ℃ of+/-, 0.22 ℃ of+/-, 0.21 ℃ of+/-, 0.2 ℃ of+/-, 0.19 ℃ of+/-, 0.18 ℃ of+/-, 0.17 ℃ of+/-, 0.16 ℃ of+/-, 0.15 ℃ of+/-, 0.14 ℃, 0.13 ℃ of+/-, 0.12 ℃ of+/-, 0.11 ℃ of+/-, 0.1 ℃ of+/-, 0.09 ℃ of+/-, 0.08 ℃ of+/-, 0.07 ℃ of+/-, 0.06 ℃ of+/-, 0.05 ℃ of+/-, 0.04 ℃ of+/-, 0.03 ℃ of+/-, 0.02 ℃ of+/-, 0.01 ℃ of+/-, 0.009 ℃ of+/-, 0.008 ℃ of+/-, 0.007 ℃ of+/-, 0.006 ℃ of+/-, 0.005 ℃ of+/-, 0.004 ℃ of+/-, 0.003 ℃ of+/-, 0.002 ℃ of+/-or+/-0.001 ℃.In another embodiment, the temperature that has basic homogeneous between liquid metal or heat-conducting fluid heat block any two slotted eyes in the heat block container holder, difference apart from ideal temperature is no more than+and/-0.6 ℃, for example be no more than+/-0.59 ℃, 0.58 ℃ of+/-, 0.57 ℃ of+/-, 0.56 ℃ of+/-, 0.55 ℃ of+/-, 0.54 ℃ of+/-, 0.53 ℃ of+/-, 0.52 ℃ of+/-, 0.51 ℃ of+/-, 0.5 ℃ of+/-, 0.49 ℃ of+/-, 0.48 ℃ of+/-, 0.47 ℃ of+/-, 0.46 ℃ of+/-, 0.45 ℃ of+/-, 0.44 ℃ of+/-, 0.43 ℃ of+/-, 0.42 ℃ of+/-, 0.41 ℃ of+/-, 0.4 ℃ of+/-, 0.39 ℃ of+/-, 0.38 ℃ of+/-, 0.37 ℃ of+/-, 0.36 ℃ of+/-, 0.35 ℃ of+/-, 0.34 ℃ of+/-, 0.32 ℃ of+/-, 0.31 ℃ of+/-, 0.3 ℃ of+/-, 0.29 ℃ of+/-, 0.28 ℃ of+/-, 0.27 ℃ of+/-, 0.26 ℃ of+/-, 0.25 ℃ of+/-, 0.24 ℃ of+/-, 0.23 ℃ of+/-, 0.22 ℃ of+/-, 0.21 ℃ of+/-, 0.2 ℃ of+/-, 0.19 ℃ of+/-, 0.18 ℃ of+/-, 0.17 ℃ of+/-, 0.16 ℃ of+/-, 0.15 ℃ of+/-, 0.14 ℃, 0.13 ℃ of+/-, 0.12 ℃ of+/-, 0.11 ℃ of+/-, 0.1 ℃ of+/-, 0.09 ℃ of+/-, 0.08 ℃ of+/-, 0.07 ℃ of+/-, 0.06 ℃ of+/-, 0.05 ℃ of+/-, 0.04 ℃ of+/-, 0.03 ℃ of+/-, 0.02 ℃ of+/-, 0.01 ℃ of+/-, 0.009 ℃ of+/-, 0.008 ℃ of+/-, 0.007 ℃ of+/-, 0.006 ℃ of+/-, 0.005 ℃ of+/-, 0.004 ℃ of+/-, 0.003 ℃ of+/-, 0.002 ℃ of+/-or+/-0.001 ℃.In another embodiment again, sample cell in liquid metal or the heat-conducting fluid heat block has uniform temperature in sample cell, difference apart from ideal temperature is no more than+and/-0.6 ℃, for example be no more than+/-0.59 ℃, 0.58 ℃ of+/-, 0.57 ℃ of+/-, 0.56 ℃ of+/-, 0.55 ℃ of+/-, 0.54 ℃ of+/-, 0.53 ℃ of+/-, 0.52 ℃ of+/-, 0.51 ℃ of+/-, 0.5 ℃ of+/-, 0.49 ℃ of+/-, 0.48 ℃ of+/-, 0.47 ℃ of+/-, 0.46 ℃ of+/-, 0.45 ℃ of+/-, 0.44 ℃ of+/-, 0.43 ℃ of+/-, 0.42 ℃ of+/-, 0.41 ℃ of+/-, 0.4 ℃ of+/-, 0.39 ℃ of+/-, 0.38 ℃ of+/-, 0.37 ℃ of+/-, 0.36 ℃ of+/-, 0.35 ℃ of+/-, 0.34 ℃ of+/-, 0.32 ℃ of+/-, 0.31 ℃ of+/-, 0.3 ℃ of+/-, 0.29 ℃ of+/-, 0.28 ℃ of+/-, 0.27 ℃ of+/-, 0.26 ℃ of+/-, 0.25 ℃ of+/-, 0.24 ℃ of+/-, 0.23 ℃ of+/-, 0.22 ℃ of+/-, 0.21 ℃ of+/-, 0.2 ℃ of+/-, 0.19 ℃ of+/-, 0.18 ℃ of+/-, 0.17 ℃ of+/-, 0.16 ℃ of+/-, 0.15 ℃ of+/-, 0.14 ℃, 0.13 ℃ of+/-, 0.12 ℃ of+/-, 0.11 ℃ of+/-, 0.1 ℃ of+/-, 0.09 ℃ of+/-, 0.08 ℃ of+/-, 0.07 ℃ of+/-, 0.06 ℃ of+/-, 0.05 ℃ of+/-, 0.04 ℃ of+/-, 0.03 ℃ of+/-, 0.02 ℃ of+/-, 0.01 ℃ of+/-, 0.009 ℃ of+/-, 0.008 ℃ of+/-, 0.007 ℃ of+/-, 0.006 ℃ of+/-, 0.005 ℃ of+/-, 0.004 ℃ of+/-, 0.003 ℃ of+/-, 0.002 ℃ of+/-or+/-0.001 ℃.

In some embodiments, the temperature homogeneity of liquid metal or heat-conducting fluid heat block is regulated by circulation liquid metal or heat-conducting fluid in heat block.The circulation of liquid metal or heat-conducting fluid can produce by natural convection or forced convection, as include but not limited to the intervention of the device of stirring rod, pump or MHD power, by material power or with exchanging or the vibration of the MHD power generation of galvanic current or the like.

In some embodiments, liquid metal or heat-conducting fluid heat block have faster than the alternating temperature speed of the speed of common metal heat block or can be to change temperature faster than the speed of common metal heat block greatly, as changing temperature with per second 5-50.5 ℃ speed at least, include but not limited to per second 10-40 ℃ scope at least, more specifically with 5 ℃ of per seconds at least, 5.5 ℃, 6 ℃, 6.5 ℃, 7 ℃, 7.5 ℃, 8 ℃, 8.5 ℃, 9 ℃, 9.5 ℃, 10 ℃, 10.5 ℃, 11 ℃, 11.5 ℃, 12 ℃, 12.5 ℃, 13 ℃, 13.5 ℃, 14 ℃, 14.5 ℃, 15 ℃, 15.5 ℃, 16 ℃, 16.5 ℃, 17 ℃, 17.5 ℃, 18 ℃, 18.5 ℃, 19 ℃, 19.5 ℃, 20 ℃, 20.5 ℃, 21 ℃, 21.5 ℃, 22 ℃, 22.5 ℃, 23 ℃, 23.5 ℃, 24 ℃, 24.5 ℃, 25 ℃, 25.5 ℃, 26 ℃, 26.5 ℃, 27 ℃, 27.5 ℃, 28 ℃, 28.5 ℃, 29 ℃, 29.5 ℃, 30 ℃, 30.5 ℃, 31 ℃, 31.5 ℃, 32 ℃, 32.5 ℃, 33 ℃, 33.5 ℃, 34 ℃, 34.5 ℃, 35 ℃, 35.5 ℃, 36 ℃, 36.5 ℃, 37 ℃, 37.5 ℃, 38 ℃, 38.5 ℃, 39 ℃, 39.5 ℃, 40 ℃, 40.5 ℃, 41 ℃, 41.5 ℃, 42 ℃, 43.5 ℃, 44 ℃, 44.5 ℃, 45 ℃, 45.5 ℃, 46 ℃, 46.5 ℃, 47 ℃, 47.5 ℃, 48 ℃, 48.5 ℃, 49 ℃, 49.5 ℃, the speed of 50 ℃ and 50.5 ℃ changes temperature.In relevant embodiment, described liquid metal or heat-conducting fluid heat block can keep the temperature of homogeneous simultaneously to change temperature than common metal heat block faster rate on whole heat block and/or in the sample in described heat block.In one embodiment, the liquid metal heat block can be with the speed elevated temperature of 44 ℃ of per seconds at least.In another embodiment, the liquid metal heat block can reduce temperature with the speed of 17 ℃ of per seconds at least.In one embodiment, the temperature of liquid metal or heat-conducting fluid is measured with granulated glass sphere thermistor (Betatherm).In another embodiment, infrared camera is used for measuring the temperature of liquid metal or heat-conducting fluid or covers the temperature of the close container holder of liquid at heat block top or the temperature of sample cell.In another embodiment, the temperature of liquid metal or heat-conducting fluid is measured with external probe.In another embodiment again, the granulated glass sphere thermocouple measurement of the temperature of liquid metal or heat-conducting fluid.In another embodiment, the temperature probe measurement of at least one sample cell.

Be used in some embodiment of thermal cycler at liquid metal or heat-conducting fluid heat block, can carry out a series of PCR circulations quickly than conventional solid heat block thermal cycler.Single simple PCR circulation generally includes denaturing step, hybridization step and extension step, and each step is carried out under specific temperature.In some embodiments, a series of 30 PCR circulations (PCR operation) can be finished for example 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes in 1-20 minute.Yet the time span of PCR operation not only depends on the speed and the temperature unity of thermal cycler.Those skilled in the art should be perfectly clear, and the time span of PCR operation may change along with the characteristic of needed PCR product.

Composition

Under the temperature of carrying out PCR be the thermally conductive material of liquid make it possible to sample cell with Any shape immerse in the temperature-control material and the thermo-contact that keeps good heat passage to carry out.Therefore, composition is a liquid in the temperature range between primer annealing and double-stranded dissociating at least preferably.Primer annealing usually occurs in about 55 ℃, but can be up to about 70 ℃ or be low to moderate about 45 ℃.Two strands is dissociated and is usually occurred in about 94 ℃, but can be according to making temperature lower as the length of guanine in the amplicon-cytosine(Cyt) base pairing and the factor of per-cent (GC content).Therefore, in one embodiment, the melting temperature (Tm) of liquid composition (i.e. transformation temperature from solid-state to liquid state) is not higher than 70 ℃.In the alternate embodiment, the melting temperature (Tm) of liquid composition is not higher than 60 ℃.In the alternate embodiment, the melting temperature (Tm) of liquid composition is not higher than 50 ℃.In another embodiment again, the melting temperature (Tm) of liquid composition is not higher than 40 ℃.

Various liquid metal compositions can be used to implement desired the present invention.In one embodiment, the liquid metal composition can be gallium or the composition that contains gallium.Some compositions also can comprise indium, copper, rhodium, silver, Ya Xi, bismuth, tin and/or zinc.In one embodiment, liquid metal from Coollaboratory (Http: //coolaboratory.com) obtain.In some embodiments, liquid metal can comprise the gallium of 40-80% and the indium of 10-50%.In some embodiments, liquid metal alloy can comprise copper, rhodium, silver, Ya Xi, bismuth, tin, zinc or its combination of 1-40%.In some embodiments, liquid metal alloy can comprise the tin, the indium of about 75% gallium/about 25%, about 95% gallium/about 5% indium or 100% gallium of indium/about 16.0% of zinc, about 62% gallium/about 22% of tin/about 1.0% of indium/about 14.0% of about 60% gallium/about 25.0%.In another embodiment, composition can comprise the gallium with the 60-99% of indium combination, according to appointment 75% gallium and about 25% indium.The alloy of about 75% gallium and about 25% indium becomes liquid and has about 2000 ℃ boiling point at about 15.7 ℃.Such alloy is in liquid state in PCR thermal cycling process, but never can reach the high temperature that is enough to vaporize.Therefore, even directly contact in the embodiment of sample cell, can not cause toxic problem owing to the vaporization of liquid metal at liquid metal yet.

In another embodiment, the present invention can use deleterious liquid metal, as mercury, mercury alloy or Wood's metal (Woods Metal).The Wood's metal that comprises about 50% bismuth, 25% lead, 12.5% tin, 12.5% cadmium has higher operating temperature range (70-350 ℃).The heat block that comprises liquid metal (as Wood's metal) can be used for heating (boil) biological sample either and be used for the laboratory technique that any other need stablize the heat piece.

In various embodiments, liquid composition is liquid metal, liquid metal alloy or the slurry that comprises metal.The coefficient of expansion of liquid metal will change according to the accurate composition that imagination is used for liquid metal, metal alloy or the slurry of thermal cycler of the present invention.In various embodiments, liquid metal, volumetric expansion about 0.1 before liquid metal alloy or metal paste begin than PCR in the PCR process, 0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9,1.0,1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8,1.9,2.0,2.1,2.2,2.3,2.4,2.5,2.6,2.7,2.8,2.9,3.0,3.1,3.2,3.3,3.4,3.5,3.6,3.7,3.8,3.9,4.0,4.1,4.2,4.3,4.4,4.5,4.6,4.7,4.8,4.9,5.0,5.1,5.2,5.3,5.4,5.5,5.6,5.7,5.8,5.9 or 6.0%.

In one embodiment, liquid metal, liquid metal alloy or the slurry that comprises metal are designed to swell increment is enough to compress joint between container holder and the sample cell, thereby form closely contact, this contact has strengthened thermal conductivity and optical transmittance (for example luminous reflectance returns in the sample cell).This contact closely by the clearance of reducing partitioned bottle seat and sample cell increased thermal conductivity between liquid metal and the sample cell.For instance, gallium has presented the expansion of suitable homogeneous.

Table 1 gallium expands

Starting temperature	End temp	Volumetric expansion
Starting temperature	End temp	Volumetric expansion	25℃	55℃	+1.08％
55℃	72℃	+0.61％	25℃	55℃	+1.08％
55℃	72℃	+0.61％	72℃	95℃	+0.83％
25℃	95℃	+2.54％	72℃	95℃	+0.83％

In another embodiment, heat block comprises the liquid composition as heat-conducting fluid.Various heat-conducting fluids can be used among the present invention.The term heat-conducting fluid comprises wax and the oil with the operating temperature range that is applicable to thermal cycler.In other words, wax and oil should have relatively low melting temperature (Tm), and scope is at 30-55 ℃, and keep their stability under the temperature of 30-110 ℃ of scope at least.Diversified wax and oil are suitable as heat-conducting fluid, comprise the compound as carbohydrate and silicon compound and composition thereof, but are not limited thereto.Example comprises: silicone oil, carboxyl modified silicone oil, mineral oil, dibutyl phthalate, poly-di-ethyl siloxane, dimethione, tetraalkoxysilane, silicon hydrocarbon (silahydrocarbons), poly-alpha olefins, naphthene base crude oil, the hydroisomerizing carburetion, paraffin oil, slack wax (parrafin wax), paraffin (paraffin wax), the paraffin of mixed nitride boron, tricosane paraffin, organopolysiloxane, polyolefin-wax, polyethylene wax or Poly Propylene Wax and composition thereof.In addition, heat-conducting fluid can comprise the additive that is used to improve its stability, viscosity, the coefficient of expansion, opaqueness and/or reflectivity.This additive comprises but is not limited to plastics, mineral aqueous fluids, antifreezing agent or metal.

Reflectivity

In one embodiment, liquid metal or heat-conducting fluid are used to reflect the light of all its receptions basically.In another embodiment, liquid metal or heat-conducting fluid be with can the excited sample groove in fluorescence molecule and the component in the heat block of the thermal cycler that is connected of the optical module of detection signal.Sample cell can directly be immersed in the liquid metal or heat-conducting fluid of heat block, or isolates with liquid metal or heat-conducting fluid by the container holder of thermal conductivity.In comprising the embodiment of container holder, container holder can be made by material substantial transparent or reflection.In these embodiments, the sample cell surface of liquid metal or heat-conducting fluid or reflection can reflected back the light of all its receptions basically, these light can be detected by optical detection device then, and optical detection device includes but not limited to CCD device, CMOS device, LED matrix, PN photorectifier, pin photodiode or photocell.In addition, in the embodiment that uses the transparent vessel seat, the transparent sample groove can use the plastics identical with container holder to constitute, and perhaps makes with having with the transparent material of the similar specific refractory power of container holder.

In one embodiment, the level of liquid metal or heat-conducting fluid will be higher than the level of sample in the sample cell.In other words, when observing from the side, the volume of sample in the sample cell and PCR reaction mixture will be lower than the level of liquid metal or heat-conducting fluid.Thereby liquid metal or heat-conducting fluid will reflect any light that enters sample cell or produce basically in sample cell.In one embodiment, liquid metal or heat-conducting fluid heat block are the parts that comprise in the real-time circulation instrument of optical module, and optical module comprises the multi-channel detection instrument, as a plurality of needle-like diodes.For example, this instrument can comprise and is exclusively used in a plurality of single passages of detection from the light in the single hole of container holder.In this embodiment, liquid metal or heat-conducting fluid play the effect that basic prevention is transferred to the detected optical buffer liquid of sense channel that light in first sample cell or that therefrom spread out of is associated with second sample cell.

Sample cell

Term " sample cell " comprises the reaction vessel of different shape and structure.In one embodiment, sample cell is used to hold reaction mixture, and for example PCR reaction mixture, reverse transcription reaction mixture, real-time PCR reactions mixture or any other need to heat, the reaction mixture of cooling or stable uniform temperature.In one embodiment, sample cell is circular or tubular container.In the alternate embodiment, sample cell is avette container.In another embodiment, sample cell is rectangle or quadrate container.Any aforesaid embodiment can further adopt tapered bottom, circular base or flat.In another embodiment again, sample cell is a kapillary, and as transparent glass kapillary or coated capillary, its floating coat (as metal) improves the internal reflection rate.In other embodiment, sample cell is a slide plate, as slide glass.In another embodiment, sample cell seals in the bottom.In one embodiment, sample cell coated (at least externally coated) anti-adhesive coating (as teflon or silane) is to reduce the adhesion of liquid composition (as liquid metal or heat-conducting fluid).In another embodiment, sample cell coated (coated in inside at least) prevents that amplicon from adhering to the material of sample cell wall (as fluoropolymer or BSA).

In one embodiment, sample cell is made into one container and uses with one container.In another embodiment, sample cell is bound up on becomes the horizontal sequence that comprises a plurality of monocyte sample grooves together, as 2,4,6,10,12,14 or 16 pipes.In another embodiment again, sample cell is bound up and is engaged in sample slotted vane, plate or dish in the heat block top of thermal cycler to form through design, thereby occupies part or all available reacting hole.In one embodiment, dish or sheet can comprise at least 6 holes, 12 holes, 24 holes, 36 holes, 48 holes, 54 holes, 60 holes, 66 holes, 72 holes, 78 holes, 84 holes, 90 holes or 96 holes, 144 holes, 192 holes, 384 holes, 768 holes or 1536 holes.

In one embodiment, sample cell has the lid that is connected to its opening end by connect elements (as optional hinged plastic strip).In another embodiment, sample cell does not have the lid of connection.In one embodiment, sample cell is configured to receive the maximum sample volume, as 10ul, 20ul, 30ul, 40ul, 50ul, 60ul, 70ul, 80ul, 90ul, 100ul, 200ul, 250ul, 500ul, 750ul, 1000ul, 1500ul, 2000ul, 5ml or 10ml.

In some embodiments, real-time polymerase chain reaction (PCR) carries out in by the sample cell of making according to the not reactive material of selecting (for example glass or plastics) of optical clarity and known and reactant.In one embodiment, sample cell is designed to make that light can enter or leave the top of sample cell from the top of sample cell, and the top of sample cell can be covered by lid that can transmitted light.In another embodiment, sample cell is designed to make light can enter or leave sample cell by the bottom of sample cell.In one embodiment, sample cell is made by transparent or translucent material that can transmitted light.In one embodiment, sample cell is designed to make light to be conducted through independent surface and leaves, such as top or bottom.

In other embodiment, sample cell is by the material of internal reflection basically, and for example reflexive plastics, coated glass plastics (as with metal or other reflecting materials), coated glass (as with metal or other reflecting materials), doped-glass (by adding the molecule manufacturing that increases the glass-reflected rate) or metal (including but not limited to stainless steel, chromium or other non-reactive basically metals) are made.Metal has high strength, make can have than glass or plastics thin the sidewall of Duoing.The thermal boundary that this has reduced between reactant and the outside hot/cold source, make better thermal control is arranged, higher room temps homogeneity and temperature variation more rapidly, all these can produce faster, more effective polymerase chain reaction.Be used for monitoring in the embodiment of real-time fluorescence at surface fluorescence, the high-reflectivity of inner metal surfaces helps the collection of fluorescence.In one embodiment, the inside of metal sample groove be polishing or electrochemical etching.

In one embodiment, the metal sample groove is made by bigger cylindrical or rectangular raw material.At first, this raw material is drawn into the ideal diameter.The standard method production that perhaps can use syringe to produce.The continuous pipe of producing (as rectangle or cylindrical tube) must be sheared and seal the fragment that has desired length with generation simultaneously, as 1cM, 2cM, 3cM, 4cM, 5cM, 6cM, 7cM, 8cM, 9cM, 10cM or any length therebetween.In one embodiment, the diameter of sealed end is no more than the diameter of pipe itself.In this embodiment, pipe is sheared subsequently and seals: pipe (1301) can be admitted in the metal block (1305) (Figure 13) by groove (1303).In one embodiment, the width of groove is in close proximity to the external diameter size of metal tube.When pipe (1301) exceeded metal block (1305), cutting blade (1307) flushed with metal block, reduces its blade perpendicular to groove.In one embodiment, blade (1307) is configured as and has taper blade (1309).In another embodiment, blade (1307) is configured as to have and cuts simultaneously and the blade of the end of closing metal tube of curling, and groove walls prevents that sealed end from evening up, thereby forces its diameter to keep approaching the diameter of pipe.After curling, sample hose may need further sealing.In one embodiment, this can utilize little welding or brazing or soldering to finish.In another embodiment, can avoid using cutting and curling by using metal closures.If necessary, last sealed end can grind to reduce diameter and to eliminate Roughen Edges.

In the alternate embodiment, the metal sample groove can be drawn to the diameter that needs and clip to the length that needs.Each segmental end is pressed in the semisphere cup with diameter similar to pipe itself then.This move can the cavetto and the closed metal pipe that curls.In relevant embodiment, the open top that latch is inserted into metal tube is beneficial to the sealed end of pipe is stamped in the reception cup.This pipe can curl sealing to produce the sample cell with a blind end and an open end that is suitable for PCR.

The sample cell lid

The top of sample cell generally wishes to be capped to prevent the evaporation in PCR thermal cycling process.This can be by forming one deck non-reactive liquid (as mineral oil) layer, sealed sample groove (as by the heat-sealing kapillary) or realizing by sealing sample cell with lid at the top of sample cell.

Used lid in one embodiment.Lid can be made with any suitable material (as glass or plastics), the feasible effect of playing vapour barrier with sample cell formation sealing.In one embodiment, lid is a plastic cover.Lid can be opaque, translucent or substantial transparent.In one embodiment, lid is optically transparent, and is applicable to thermal cycler and the optical module that comprises liquid metal or heat-conducting fluid heat block.In another embodiment, cap portion ground or all be coated with opaque coating.In another embodiment again, cap portion ground or all be coated with reflective coating, for example mirror coating.

From sample cell or from include with excitation wavelength fill can the time extract light the fluorescigenic container of material and may be difficult to finish, especially when heat block covers this container.Make and the sample cell lid (303 that randomly comprise light guide with material transparent (as plastics or glass); 403) can (scioptics, wave filter, beam splitter are as Fig. 3) couple light to light source (as optical transmitting set) and detector, wherein the existence of light guide has increased the amount of the light that is transmitted to detector.Optical transmitting set can be coherent source (as laser apparatus) or incoherent source (as LED).

In addition, the advantage of an exemplary embodiment of the present invention is to be reflected back toward in the sample cell by the light of the marker in the sample cell/dye sample emission reflection selectivity based on liquid metal or heat-conducting fluid, liquid metal alloy or metal slurry.In further embodiment, volume reflection is by (for example forming better contact between sample cell and container holder slotted eye, utilize volumetric expansion to form closely and engage, thereby perhaps on the wall of container holder slotted eye, apply extra pressure to form tight joint with sample cell by the pressure liquid that uses pump to increase the chamber interior that comprises liquid) be further enhanced.

In another embodiment, can realize similar luminous reflectance effect by utilizing special sample cell of the present invention, wherein sample cell is made up of opaque composition (for example stainless steel) or (inner/outer) is coated with reflexive opaque coating (for example aluminium).In one embodiment, sample cell is a mirror coating.Comprise in the embodiment of metal at sample cell, metal can polish or electrochemical etching to increase its reflectivity.

In either case, in various embodiments, lid utensil of the present invention is made up of optically transparent material, and its characteristics have low autofluorescence or do not have autofluorescence, and forms excellent sealing with sample cell.In addition, in some embodiments, lid forms the projection (for example light guide) of outstanding certain-length in the sample cell.The examples of material that can be used to form lid includes but not limited to acrylic resin, polycarbonate, polystyrene, styrene block copolymer (SBCs), styrene-acrylonitrile (SAN), ABS, polysulfones, thermoplastic polyester (as PET), polypropylene, vinylformic acid styrol copolymer (SMMA), polyvinyl chloride, nylon, celluosic resin, cyclic olefine copolymer (COC), allyl diglycol carbonates (ADC), cycloolefin is (as TOPAS, ZEONOR and ZEONEX) and composition thereof.

In various embodiments, lid and/or projection can be to include but not limited to Polygons, ellipse, circle, square, rectangle, leg-of-mutton geometrical shape, or any shape by injection moulding (as known in the art) acquisition.In addition, can recognize that sample cell and container holder wall also can be the shape of any needs.In one embodiment, lid, sample cell and container holder are identical geometrical shapies.In further embodiment, geometrical shape is a rectangle.In one embodiment, geometrical shape based on lid, sample cell and container holder wall each material that uses select, strengthening the contact between sample cell wall and the container holder wall, and provide best to the outer light transmission of sample cell.More further in the embodiment, the specific refraction of sample cell wall and container holder wall equates or is almost equal.

In one embodiment, lid and lid projection are made up of identical materials.In one embodiment, each is made up of lid, lid projection, sample cell and container holder identical materials or different materials, and these materials at this openly.

In some embodiments, the lid projection be projected into that the level (as line) at liquid metal in the sample cell or heat-conducting fluid, liquid metal alloy or liquid metal slurry (being referred to as " liquid ") place is above, this level place or this level below horizontal.

In one embodiment, light will be sent in the sample cell by projection, and the signal by the marker in the sample cell/dye sample emission that is obtained is helped to transmit by projection; This light emits from the discrete basically part of lid, is detected by photoelectric detector (for example photorectifier (as Fig. 6)) herein.

In one embodiment, the lid tip is lower than the liquid level of liquid metal or heat-conducting fluid.If observe from the side, the lid tip clearly is lower than the level of the liquid metal or the heat-conducting fluid at sample cell place, thereby it collects all basically light.This is to be used for sample cell directly to be immersed in the embodiment of liquid metal or heat-conducting fluid and the situation that sample cell places the embodiment of container holder slotted eye.

The shape of lid and surface quality can be designed in such a way that the optically-coupled maximization from the lid to liquid.In another embodiment, lid can be with fibre bundle or by the irradiation of freeboard scheme.The freeboard system can comprise coupled lens and/or beam splitter.Liquid and detection fluorescence but the optical system simultaneous excitation fluoresces.

In one embodiment, plastic cover can be by casting plastics or injection moulding manufacturing.

In one embodiment, lid absorbs at an easy rate from the heat of thermal cycler/liquid emission, so lid is heated to the temperature that reduces or eliminate condensation.Usually, use the PCR method of prior art, upper area/the lid of sample cell will have lower temperature than the bottom of sample cell, therefore, thereby reaction liquid will condense to the variation (for example, the variation of concentration causes the reaction or the wrong result of poor efficiency) that causes the chemical property of reacting on the colder surface.In another embodiment, sample cell is designed to make sample cell to stretch enough far under fluid level, thus the top of sample cell inlet enough away from the surface of sample/mixed solution to prevent condensation.In another embodiment again, the downward pressure on top cover or the lid forces sample cell to form with container holder and contact more closely, and this can postpone to evaporate.

The device of heating and cooling composition

Liquid metal or heat-conducting fluid heat block can come heating and cooling by using various technology well known by persons skilled in the art.In one embodiment, the heating unit of heat block is selected from amber ear card device, resistance heater and radiation heater.In one embodiment, the heat block cooling element is selected from amber ear card device, scatterer, refrigerator, evaporative cooler, heat pipe, heat pump and phase change material.In one embodiment, heating unit can provide by pipe is extended in the heat block.Pipe can be equipped with pumps hot or cold fluidic pump.In some alternate embodiments, liquid metal or heat-conducting fluid heat block can be equipped with heating and/or cooling coil, or are equipped with the resistance heater that prevents fringing effect.In another embodiment, liquid metal or heat-conducting fluid heat block can be thermally coupled to the peltier effect thermounit.

In one embodiment, heat block is designed to make liquid metal or heat-conducting fluid to keep the temperature of homogeneous on whole.

The multizone heating unit

In one embodiment, liquid metal or heat-conducting fluid heat block are thermally coupled to heating unit, as multizone heating and biasing cooling system.In one embodiment, the biasing cooling system provides little constant refrigerative coolant flow by bottom or lateral biasing cooling channel attached to heat block.The little thermosteresis of constant that this has caused heat block compensates this thermosteresis by the multizone well heater.Well heater be used to hatch segmental sample blocks thermal coupling, wherein the temperature of sample blocks remains on stable value.The little thermosteresis of constant that is caused by the biasing cooling system makes Controlling System to carry out the ratio control that temperature rises or descends with little increment.This means and to carry out the heating and cooling of controlled, foreseeable, little ratio to correct the temperature mistake of heat block by temperature controlling system.The multizone well heater can be controlled by CPU.

In one embodiment, heating unit is formed (Figure 10) by one or more kind of thread elements such as heater coil.This kind of thread elements is arranged as to be provided best heating and reduces or eliminates fringing effect.For instance, kind of thread elements is arranged on the periphery of heat block.In addition, using under the situation of a plurality of kind of thread elements, they are equidistantly arranged, perhaps internally to peripheral with increase/minimizing apart from gradient array with heating that the best is provided and reduce or eliminate fringing effect.In one embodiment, kind of thread elements is arranged in the bottom of piece with checker board pattern.In the alternate embodiment, kind of thread elements is with concentric(al) circles or the oval bottom that is arranged in heat block.In another embodiment, kind of thread elements is arranged along the bottom and the side of heat block.In another embodiment again, kind of thread elements for example hangs, piles up or comprise the three-dimensional arrangement of more than one chessboard layer so that liquid metal is arranged around the form of all side flow of kind of thread elements.In another embodiment again, kind of thread elements more near the edge section of heat block than at the interior location of heat block more near (as variable spacing), with the outward flange of offsetting heat block with than inside speed of cooling refrigerative tendency (Figure 10) faster.Be known in the art, fringing effect causes the inconsistent of temperature homogeneity, normally at the outer peripheral portion of heating unit.

In one embodiment, coolant control system passes through through the continuous hydronic liquid coolant in the biasing cooling channel that the input and output pipe links to each other with heat block (for example mixture of automobile antifreeze solution and water).Coolant control system is also controlled and is passed through the more liquid stream of the even change cooling liquid flowing approach of heavy body in the heat block.The even but passage of turning cold is used for changing apace by the heat block of flowing through with the refrigerative liquid coolant of high relatively flow velocity pumping large volume the temperature of heat block.

In one embodiment, the liquid coolant that is used to cool off heat block mainly is made up of water and ethylene glycol mixture.Liquid coolant is that the heat exchanger by the receiving fluids refrigerant comes refrigerative, and it extracts heat by input tube from sample blocks.Heat exchanger receives pressurized liquid refrigerant (as freonll-11 or ethanol) by input tube from refrigeration unit.This refrigeration unit generally comprises compressor, fan and finned tube heat radiator.The refrigeration agent that the refrigeration unit compression receives from heat exchanger.The refrigeration agent of heating is cooled in finned tube condenser and is condensed into liquid.The pressure of refrigeration agent is kept above its vapour pressure by the flow restrictor kapillary in finned tube condenser.The input of this output capillaceous and heat exchange device is coupled.In heat exchanger, the pressure of refrigeration agent allows to be reduced to it below vapour pressure, makes its expansion.In this expansible process, the liquid coolant that heats up from round-robin heat exchanger absorbs heat, thereby and this heat transferred refrigeration agent cause the boiling of refrigeration agent.The refrigeration agent that heats up is discharged from heat exchanger and compression then, and circulates by finned tube condenser once more.Fan is blown into air by finned tube condenser, causes exchanging from heat in the refrigeration agent of pipe and ambient air.In one embodiment, process of refrigeration can extract at least 400 watts heat from liquid coolant in the time of 30 ℃, can extract at least 100 watts heat from liquid coolant in the time of 10 ℃, to support temperature cycle fast.

At an embodiment, the biasing cooling can be cancelled, and is perhaps provided by other devices, as engaging or normal recirculated water (as distilled water or tap water) by use cooling fan and the cooling fins that forms in the sample blocks metal, amber ear card.

The peltier effect thermounit

Amber ear card device or element are also referred to as thermoelectricity (TE) module, are the little solid-state devices with heat pump functional.Typical amber ear card unit is several mm thick and several millimeters to several centimetres squares.It is by two ceramic plates and small-sized Tellurobismuthite (Bi therebetween ₂Te ₃) sandwich structure that forms of cubical array.When applying direct current, heat is transferred to opposite side from a side of device, can remove by scatterer at this heat." cold " side can be used for cooling electronic device, as microprocessor or photoelectric detector.If current reversal, device just changes the direction that heat moves.Amber ear card device does not have moving-member, need not refrigeration agent, does not produce noise or vibration, and volume is little, and the life-span is long, and can realize precise dose control.Can carry out temperature control by use temperature sensor feedback (as thermistor or solid state sensor) and closed control circuit, this control can be based on general purpose programmable computer.

In the alternate embodiment, thermal cycler comprises liquid metal or the heat-conducting fluid heat block with heating unit (it is Peltier's element) thermal coupling, to obtain ideal temperature distribution (temperature curve in the section at the appointed time) (Fig. 1-4) in liquid metal or heat-conducting fluid heat block.In this embodiment, depend on the temperature that needs obtain, Peltier's element is used as the element of cooling or heating in a temperature distribution.

In another embodiment, thermal cycler can further comprise resistance heater and the Peltier's element that is used in combination, to obtain the accuracy and the consistence of needed temperature changing speed and needed temperature distribution in liquid metal or heat-conducting fluid heat block.

In one embodiment, thermal cycler comprises at least one Peltier's element, the thermal cycler parts that its temperature cycle that forms the heat block that is used to comprise liquid composition changes.At least one heating surface of Peltier's element and liquid metal, heat block plate or radiator form large-area thermo-contact, and other heating surfaces form large-area the contact with the cooling element that is used to dispel the heat.Cooling element can be a metal, for example aluminium or copper.Thermal cycler can further comprise the fan that is used to dispel the heat, but it randomly is a switch.In the alternate embodiment, liquid metal or heat-conducting fluid heat block combine with Peltier's element and form discrete unit (swap block), and it can be removed from the thermal cycler main body.Swap block can further comprise and Peltier's element coupled scatterer and/or fan.This swap block can be removed from thermal cycler with place under repair or with the swap block with difference in functionality element and replace, and is designed to hold the sample cell of different sizes with previous swap block and shape as container holder.Further along with development of technology, can upgrade swap block and need not to buy brand-new thermal cycler.

In another embodiment, comprise the heat block of liquid composition and a plurality of Peltier's element thermal couplings on first side that is positioned at described heat block located adjacent one another.In the alternate embodiment, comprise liquid composition (501; 551; 571) heat block and a plurality of amber ear card device (503; 553; 573) thermal coupling, wherein at least one amber ear card device is positioned on first side of described storage vault, and at least the second amber ear card device is positioned on second side of described storage vault.In one embodiment, has one at least from commercial source (as Marlow Industrries or Nextreme Thermal Solutions) amber ear card device that obtains and heat block thermal coupling (the Nextreme Thermal Solutions that comprises fluid cpds; 3040 Cornwallis Road, P.O.Box 13981, Research Triangle Park, NC 27709-39810).

In one embodiment, make Peltier's element avoid the influence of thermodynamics mechanical tension peak value by the central spring stationary installation of pushing Peltier's element and keep Peltier's element and heat block to lean on mutually.Peltier's element can flexibly be interposed between the heating surface and cooling element of heat block.The surface in contact of cooling element can be pressed against on the Peltier's element by for example pressure spring or similar device.In one embodiment, the tension force of spring can pass through screw, spring washer and ball and socket joint to be regulated, and it can further improve the degree of freedom of cooling element.

In the alternate embodiment, Peltier's element is specially as refrigeration (heat extraction) element.In other words, it only is used to cool off heater unit.This will prolong the work-ing life of Peltier's element.

In another embodiment, thermal cycler can be integrated and be positioned around the heat block and along the peripheral localized resistance heater of heat block outer wall.In this embodiment, Peltier's element can only be used for cooling.This makes Peltier's element alleviate the influence of mechanical heating power, thereby helps to prolong the work-ing life of the Peltier's element in the thermal cycler.

Top cover

In some embodiments, liquid metal or heat-conducting fluid heat block are parts that randomly comprises the thermal cycler of plug (as hinged top lid).In various embodiments, stopper can pass through various devices (for example, clip, spring, screw etc.) and be fixed on the piece.In one embodiment, stopper comprises sealing and the squeezing device that is used for fixing the sealed sample groove in the container holder that is positioned at liquid metal or heat-conducting fluid heat block.In the alternate embodiment, sealed sample groove when stopper is closed.Stopper can have spring and keep pressing plate, and it is pressed into each sample cell in the container holder slotted eye of liquid metal or heat-conducting fluid heat block with certain strength.Stopper can comprise further that the top cover that is used for the lid shape remains on the recess of sample cell and/or is used for the opening that the liquid-transfering needle with the co-axial pressing plate of sample cell penetrates.Spring element can comprise wave washer.In one embodiment, safety ring prevents that pressing plate drops when hinged top lid is opened.

In another embodiment, stopper comprises heating unit.In the alternate embodiment, the stopper of thermal cycler has been integrated the feeler mechanism that can detect light.This stopper can further comprise heating unit.For some analyses, as PCR, it is necessary making container holder be warmed up to controlled temperature.At high temperature, the content of sample cell tends to higher speed evaporation.Therefore in order to reduce evaporation and to cause material to lose from container holder, container holder can cover with the coverture of the temperature that is heated to above the sample in the sample cell that container holder comprises.In one embodiment, obducent temperature is heated to higher 5 ℃ than the temperature of sample cell at least.

In one embodiment, the top cover of heating comprises the coverture with the size that is used to cover the container holder top.The thin plate that lid has at least two inflexible, made by heat-stable material (as pottery, glass or silicon rubber).Being interposed between two plates is resistance heating element, and it can be the form of minor diameter nichrome wire or form by deposit resistive material (as nichrome or tin protoxide) on a plate of described plate in one embodiment.For example, No. 36 nichrome wire with 12 ohm of every foot resistivity can be used for providing enough heats to coverture.This heating unit can be configured to the serpentine form on whole plate zone, so that the heating of homogeneous to be provided on whole coverture.Can be used to space between retaining plate and the infill panel as the packing material of Resins, epoxy.

In relevant embodiment, heating unit is connected on the variable power supply, and it can be controlled to provide and add the electric current of thermal covering to the ideal temperature.Lead-in wire between power supply and the heating unit can be flexible and be configured to avoid going between in stress, thereby coverture can move without restriction, for example, is moving by the robot device in the cut-and-try work station automatically.On coverture, can provide temperature sensor to measure its temperature and to provide the control power supply to obtain the feedback of ideal temperature.

In the alternate embodiment, imagination is lower than the situation of ambient temperature for the temperature of sample cell, may it is desirable to cool off that obducent temperature is lower than surrounding environment but the temperature that is higher than material steam.This is in order to keep the minimum temperature difference between coverture and the material, thereby obducent temperature can not have influence on the controlled temperature of material in the container holder.

Optical module

In all respects of the present invention, device of the present invention is configured to provide the device of measuring the interior marker surveyed of the sample cell comprise reaction (as, PCR in real time).Each embodiment and the detection optical parts of device of the present invention are compatible fully, thereby can finish quick nucleic acid amplification/detection.The signal reflex rate of thermal cycler of the present invention by the temperature of homogeneous being provided in PCR or correlated process, relenting and increase fast can make amplification, detects and measure more accurate.Thus, an advantage is that thermal cycler of the present invention can use with the optical module of price economy, and still obtains equally accurately or more accurate real-time measurement.Therefore, thermal cycler of the present invention can utilize cheap optics and conventional optical module to realize quick, accurate and reliable DNA cloning and detection.

Optical measuring device is known in this area.In general, measure optics and comprise light source, for example it is formed by photodiode.Light source directive measured zone (sample cell/pipe).And this light source is controlled by the Evaluation and Control circuit that can be connected with the computer operation that comprises the computer executable program that is used to control optical measurement.Measure optics and also comprise the detector that can receive light from whole measured zone.This detector links to each other with evaluation circuit/computer.

The optical module that can be used for various embodiments of the present invention comprises the assembly that contains CCD imager, cmos imaging instrument, line scanner, at least one photorectifier, at least one phototransistor, at least one photomultiplier cell, at least one avalanche photodide, microlaser or Q-Q-swith laser Q.In various embodiments, according to the difference of signal from sample hose to be detected, readout device can be that reflection, transmission, surface fluorescence/fluorescence, chemical-biological are luminous, magnetic or amperometry reader (perhaps two or more combinations), needle-like diode or other readout device as known in the art.

In various embodiments, can be coupled to light source in the optical module of the present invention and comprise photodiode, laser diode, VCSEL, VECSEL, DPSS laser apparatus or can be connected with light source (as big Optical Maser System, laser diode or lamp) coupled optical fiber subsequently.In another embodiment, described diode is a laser diode.Laser diode can be used to illumination, and photodiode detector has splendid sensitivity, and most of material has minimum autofluorescence in relevant SPECTRAL REGION.Can use the LED or the photorectifier of any routine, for example, the pin photodiode that obtains from Pacific Silicon Sensors (5700 Corsa Avenue, WestlakeVillage CA, 91362).

In various embodiments of the present invention, optical module is configured to provide as 365/460,470/510,530/555,585/610,625/660, the wavelength of 680/712nm excite (light source)/emission (detector).In some embodiments, use in addition ,+-the various filter windows of 5-20nm.Various fluorescence filter groups are commercially available (as, Omega Filters; Omega Optical, Inc or INTORR), comprise the dyestuff specificity wave filter that is used for single or multiple labeling fluorescence.

In some embodiments, the light source with the specification that provides in the table 1 is provided optical module:

Fig. 6 provides an example of optical module, and wherein pin photodiode is a fluorescence detector.For example, the light source 603 that provides by the light of excitation filter 605 is provided the optical module that is used to detect fluorescence, and this light is guided by dichroic reflector/reflective mirror 611, enters in the sample cell by the object lens 609 that light beam focused in the sample cell.Same lens are collected by sample composition (as, the green or Cy5 of SYBR; Intercalative dye disclosed herein) fluorescence of Chan Shenging, this emission light is directed in the detector module pin photodiode 617 by two tropism's spectral filters 611 and by means of barrier filters 613, condenser lens 615.Output is digitized and is shown as electrical patterns or is presented on the paper or is recorded 619.In addition, the use of surface fluorescence can have extra advantage, promptly is not subjected to the restriction of size, and like this, the lens of numerical aperture aspheric surface or ball-type can be used as the collection optics.

In further embodiment, extra excitation filter can be set to be used to increase the spectrum adjustment.In one embodiment, light source is Cree XLamp, and fluorescence detector is T5 or T18 needle-like diode.

In one embodiment, optical module is by a same part (for example lid) emission and the detection light of sample cell.In another embodiment, the optical module light source is launched light (as to the PCR reactant) from a part of sample cell, and from another part of sample cell detect light emission (for example, light source in the bottom and detector at the top, vice versa).

In one embodiment, readout device is the LED readout device that detects fluorescent signal.Fluorescent signal is excited by emitting light emitting diode in SPECTRAL REGION and in the absorption peak of signal (for example fluorescent mark).Institute's fluorescent signal emitted is detected by photorectifier.In addition, the wavelength of the detection signal broadband filter that can use blocking-up spurious emission light and transmission to have the light of the peak emission wavelength of fluorescent emission mark or its above wavelength limits.In other embodiments, broadband filter can be replaced by bandpass filter.In addition, exciting light can limit by bandpass filter.

In some embodiments, excitation light source and detector are installed on the single mechanism (as the thermal cycler main body), are molded as piece (shown in Fig. 1-2), to simplify reading of the fluorescent signal that produces in the sample hose.

For example, Cy5 is the fluor that glows with very high optical extinction coefficient commonly used.The common form of this type of fluor comprises its N-hydroxy-succinamide ester or related dye Cy5.5.These dyestuffs are indoles two carbon cyanine type dyes, are normally used for flow cytometry and automatic fluorescence sequenator, and can (Pittsburg Pa.) obtains from Amersham.The amidites that Cy5 can be used as directly, is incorporated into automatically in the oligonucleotide is commercially available.In one embodiment, adopt device of the present invention to handle the sample of using the Cy5 mark.For example, when the optical element of selecting instrument to use, spectrographic red/region of ultra-red work is favourable.

In another example, the real-time measurement of pcr amplification can utilize SYBR green.But, should be noted that in some embodiments, any in various marker/dyestuffs known in the art and disclosed herein can be used for thermal cycler device of the present invention (for example, fluorescence green, quantum dot etc.) as required.

In various embodiments of the present invention, the system that comprises circulation instrument of the present invention also comprises the set of photoelectric detector, and wherein each detector provides output signal.This system also comprises at least one light source.The porous plate (305 that the position of light source is passed the light of being launched to remain on sample cell; 405) or in the band or on the contrary by the porous plate (305 of sample cell; 405) or the corresponding slotted eye that provides of band, and be sent to the set of corresponding photoelectric detector or photoelectric detector.This system comprises that also treater or other are used to analyze the device from the signal of a plurality of detector outputs.In the alternate embodiment, device of the present invention does not need to use expensive fluorescence dye or dyestuff mark, and it needs large-scale integrated optical source and detection optical parts in the system.For example, integrated real-time PCR system may spend 90000 dollars (Applied Biosystems ABIPRISM.RTM, 7700 sequence detection systems), 7500 dollars of non real-time in contrast to this PCR detection system (GeneAmp 9700) costs.Two systems all carry out the pcr amplification of 96 orifice plates, but GeneAmp 9700 needs independent spectrophotometer or fluorescence orifice plate reader to carry out the DNA measurement of concetration.

Because the footprint of LED or laser diode is very little, a plurality of LED or laser apparatus with different wave length can be incorporated in the individual packaging, and perhaps several LED/ laser apparatus in groups can very closely be separated placement to excite a hole/sample hose.

In some embodiments, in fact on behalf of several, LED have different wave length but very near at interval LED.Therefore, in further embodiment, detector also carries out similar configuration.

In some embodiments, the light source that is used for optical arrangement of the present invention can be LED or laser diode.Other light sources also can use.In addition, the number that is used for the light source relevant with each slotted eye can change.In a unrestriced example that uses device of the present invention, LED is activated to launch the light of first wavelength or first group of wavelength, and dyestuff in this optical excitation sample or marker are to transmit.This signal is detected by suitable detector, and then or simultaneously the 2nd LED pipe is activated, and its emission has the wavelength different with a LED or the light of set of wavelengths.Different dyes or marker in this optical excitation sample are to launch the signal that is detected by suitable detector.The 2nd LED closes, and then or simultaneously the 3rd LED is activated.The 3rd LED emission has the wavelength different with first and second LED or the light of set of wavelengths.Different dyes or marker in this optical excitation sample, the signal that its emission is detected by suitable detector.These measurements are very rapidly carried out and by Computer Processing, are produced demonstration.The set of the light source of tight spacing can be configured to sequentially launches light as mentioned above.

In other embodiments, different light sources are configured in the optical module, and utilization of optical detector detects independently, wherein detect and carry out at one time.

In one embodiment, device of the present invention disposes light source and array of detectors.Be appreciated that array of source comprises a plurality of light sources that are positioned on suitable substrate or the installation component and arrange by specific pattern, normally grid type.The slotted eye array comprises a plurality of sample slotted eyes (Fig. 1,2), also supports and be positioned on the suitable maintenance substrate or wherein.Photodetector array comprises a plurality of photoelectric detectors of installing similarly and arranging, to receive by sample cell (305; The light of 405) array emission, for example lid (303 by sample cell; 403) (Fig. 3).

In some embodiments, the rail system (rail system) that comprises a plurality of independently light sources (having identical or a plurality of different wavelength) can be configured on the row sample with the arrangement mode of the multirow detector with respective number.For example, the number of light source and relevant detection device can be 8 arbitrary sources that possible have a plurality of wavelength, and 8 different detector means that can be detected a plurality of wavelength of excited sample emission respectively detect.Therefore, this rail system can be operated the continuation column of the sample cell of detecting to move around.Can use various commercial wave filters as mentioned above.In one embodiment, detector comprises the optics of making cheaply by injection moulding.

In some embodiments, can use high-capacity LED.Also can use avalanche photodide, for example silicon carbide, gan, gallium arsenide or silicon or the blue silicon photoelectric diode that strengthens.The sensitivity of avalanche photodide is higher than simple photodiode far away, because signal has been amplified by avalanche process.Avalanche photodide has the typical gain between 10 to 10000, this means that signal has been exaggerated 10 to 10000 times.The improved measuring technology that use resembles locking-in amplifier also can enlarge the dynamicrange of measurement.

In one embodiment, the PCR system utilizes light source, and particularly LED is used for the emission of this light, and new possibility and application is provided.In one embodiment, led light source is contemplated to the ultraviolet LED based on gan.In another embodiment, light source is cree xlamp750 milliwatt superbright LED.

In one embodiment, use single wavelength emission.Do not need extra grating or wave filter or complex optical parts to remove to select required wavelength or carry out light to focus on.This has simplified experimental installation greatly, and has reduced the cost of assembly.Another advantage is that the cost of LED is lower.Semiconductor LED can be mass-produced, and manufacturing cost is extremely low.At present the cost based on purple, blueness and each packing plant of green LED of GaN is about 10 to 50 cents, and the price based on the LED of GaN of following scale operation is estimated in similar scope.Multi-wavelength LED can be integrated into the mixing led chip, maybe can develop special LED, and its emission can be switched between two or more wavelength.

Another advantage relates to the high-output power of LED again.The LED of output rating level in the 10mW scope can realize in the near future.In addition, the typical footprint of LED is 200um * 200um, this means that light intensity can focus on than the zonule and reduced the amount that need be used for making the extra optics (for example lens) that light concentrates.This high-throughput PCR system that has a large amount of slotted eyes (48,96,384 or 1536 hole) and correspondingly have a less slotted eye for every plate is sizable advantage.Even have 48,96 or 384 slotted eyes, the slotted eye size is still even as big as cooperating several LED in the single slotted eye zone of slot orifice plate.

In addition, another advantage relates to the array of LED.A plurality of LED also can be arranged in the led array of one dimension or two dimension.Make it possible in a plurality of slotted eyes, carry out simultaneously fluorescence measurement like this.A large amount of parallel processing has obviously reduced Measuring Time and has improved treatment capacity.This may be the important cost/time factor in the PCR system operation, is particularly tending under the trend of high-density slotted eye more.

In addition, another advantage of LED is that these light sources can be pulses.For fear of the fading or heat of dna molecular, the LED photodiode can produce short and strong pulse.LED can be on very short time scale (about 1 nanosecond nanosecond to 100) switch, this depends on design, size and the encapsulation of LED.The pulse of LED also can utilize common lock-in techniques and be synchronous with the photoelectric detector reading, to obtain better signal/noise ratio and higher sensitivity range.In addition, LED before obtaining stable light output without any need for warm up time.This deuterium and xenon lamp with the warm up time that needs several minutes before steady running at least is opposite.

Another advantage again of using the LED illuminating source is the long relatively work-ing life of this class component.The operation life that can believe LED was 10000 hours or longer.Use this light source in most systems, can avoid changing LED.

In one embodiment, provide the utilization fluoroscopic examination to carry out the method that the polymerase chain reaction is analyzed.This method comprises: the photoelectric detector that comprises porous plate, thermal cycler, output signal is provided is provided, orientates as and make light by the porous plate body and arrive at least one light source of photoelectric detector and be used to analyze the system of device of the output signal of photoelectric detector.This method comprises that also acquisition will carry out the sample that the polymerase chain reaction is analyzed.In addition, this method comprises sample setup in porous plate.And this method is included in carries out the polymerase chain reaction in the sample.This method also comprises from source emissioning light so that light passes sample arrival photoelectric detector.And this method comprises that the output of analyzing photoelectric detector is with the specific absorption of determining emitted fluorescence and the information of indicating the polymerase chain reaction.

In another embodiment, in thermal cycler, carry out PCR after, the sample emitted fluorescence is carried out rough detection, this thermal cycler comprises the heat block that comprises liquid composition.In one embodiment, the amount of rough detection non-quantitative amplified production or amplicon, but only detect whether there is described amplified production.Rough detection can be carried out with cheap optics or detector, seldom or not need to need wave filter and/or plastic lens.In one embodiment, only use photorectifier or LED detector.In another embodiment, detect fluorescence with the reactive filter system, as time resolved fluorescence (TRF).On the one hand, light source (LED or laser diode) is closed, and fluorescence molecule is short but continue luminously in measurable time, and the light of LED/ laser apparatus can be measured rather than must be filtered out to the light of these emissions.

In another embodiment again, detection system adopts the probe based on FRET.In one embodiment, detection system can almost detect two kinds of different wavelength simultaneously.Utilize the FRET probe, two fluorophores are positioned on the probe.The single excitation light source excites of sample that comprises probe.This light source has the wavelength that excites first fluorophore, and first fluorophore is luminous, and wherein most of light is absorbed by the second contiguous fluorophore.Second fluorophore is luminous with the wavelength that is different from first fluorophore.These two signals are all detected subsequently.

According to this illustrative embodiments more on the other hand, provide and carried out the method that the polymerase chain reaction is analyzed.This method is based on uses light as described below.This method comprises: the system that comprises with lower member is provided: (i) in order to keep the porous plate of a plurality of samples, (ii) thermal cycler, the photoelectric detector of output signal (iii) is provided, (iv) a plurality of light sources, its position can make the light of emission pass porous plate and arrive photoelectric detector, and (v) is used for analyzing the device of the output signal of photoelectric detector by detecting UV-light.This method comprises that also acquisition will carry out the sample that the polymerase chain reaction is analyzed.In addition, this method also comprises sample setup in porous plate.This method also is included in carries out the polymerase chain reaction in the sample.This method also comprises from a plurality of source emissioning lights so that light passes sample arrival photoelectric detector.This method also comprises the output signal of analyzing photoelectric detector.

In various embodiments, the ON/OFF time of LED is normally about several nanoseconds or tens nanoseconds.In other embodiments, laser diode even faster has switching time of subnanosecond.

In the alternate embodiment, the PCR system and correlation analysis and the technology that do not need to use fluorophore detection light source relevant and optics are provided with other.For example, LED can comprise the LED of 260 nanometers and 280 nanometers.For carrying out absorbance measuring, at first open the LED of 260 nanometers, and detect the light that absorbs in 260 nanometers by photoelectric detector.Close the LED of 260 nanometers, open the LED of 280 nanometers and the light that absorbs in 280 nanometers by the photoelectric detector detection then.These measurements can very rapidly be carried out one by one, because they do not need sample to carry out any physical motion.LED opening and closing very apace.Therefore, in an alternate embodiment, Optical devices can save the fluorescent primer that adopts in many real-time PCR systems and the expense of Taqman probe.

In one embodiment, the cheap ultraviolet LED array combination of the light of emission 260nm and 280 wavelength is in the PCR system that has with the slot orifice plate form of liquid medium (as liquid metal or hot-fluid) (directly/indirect) thermo-contact.Projector and array of detectors have the consistent configuration of arrangement of the sample cell in the liquid composition heat block of possibility and thermal cycler.For example, be similar to sample cell (305,405) among Fig. 3.Therefore, a plurality of ultra-violet light-emittings unit is located (Fig. 1) as LED with respect to a plurality of slotted eyes, thereby passes through corresponding slotted eye (and the sample that wherein comprises) from the light of each LED emission, and is received by corresponding photosensitive device or photoelectric detector.Can use optical filter, perhaps place the input terminus of photoelectric detector, perhaps place the output terminal of LED/ laser apparatus, or place aforementioned two to sentence any unwanted light of inhibition (as the light beyond the centre wavelength peak value of LED emission) (Fig. 3) simultaneously.For example, sometimes LED since in the LED active region reorganization of the reorganization of defective or the carrier the LED active region outside with long wavelength demonstration fluorescence.These unwanted fluorescence are usually less than 3 to 4 grades of main fluorescence peaks.However, also may need to utilize optical band pass filter that this fluorescence is further suppressed.

In various embodiments, optical module well known in the prior art system can be configured to and is used for thermal cycler device of the present invention.This optical module and the example of reagent that is used for the detection reaction product are at U.S. Patent application the 2006/0134644th, 2006/0014200,2005/0255516,2005/0237524,2005/0136448,2004/0133724,2003/0059822, No. 2002/0034746, and United States Patent (USP) the 7101509th, 6942971,6940598,6911327,6783934,6713297,6403037,6369893,7122799,7113624,6037130,5792610,5440388,6873417,6998598,6437345, open in 53011059 and No. 6388799, more than each is openly introduced at this by reference in full.

Control unit

In various embodiments, control unit and thermal cycler of the present invention are operatively connected.For example, such control unit comprises programmable calculator, but this programmable calculator comprises the computer actuating logic of the either side that is used to operate apparatus of the present invention, method and/or system.For example, control unit can the On/Off electric motor, fan, heating unit, stirring rod, Continuous Flow device and optical module.Control unit can be programmed to handle automatically sample, a plurality of PCR of operation circulation, obtains measuring result, with measuring result be digitized as data, with data-switching one-tenth figure/table and report.

In order to monitoring instrument operation, recording signal, processing and analytical signal or data computing machine can be PC (PC), digital machine, the treatment unit of computer, portable computer or other type based on microprocessor any.In general, computer comprises central processing unit, uses machinable medium to put down in writing and read storage or mnemon, communicating terminal (as wired communication device or wireless communication apparatus), take-off equipment (as display terminal) and the input unit (as keyboard) of information and program.Display terminal can be a touch-screen display, and in this case, it can be used as display unit and input unit simultaneously.Can there be different or additional input unit, pointing device (as mouse or control stick) for example, and can have different or additional take-off equipment, for example rattler (as loud speaker), second indicating meter or printer.Computer can move any in the several operation systems, for instance, such as each version of Windows, perhaps each version of MacOS, perhaps each version of each version of Unix or Linux.

In some embodiments, control unit carry out necessary programs with will from reaction vessel detect and measure signal digitalized, and data processing become readable form (for example form, figure, grid, graphic representation or other output form well known in the prior art).This form can be shown or record by electronics, perhaps provide with paper-based form.

In some embodiments, the circuit that control unit control is connected with heating unit, thereby the circulating temperature of adjusting/control thermal cycler of the present invention.

In further embodiment, control unit produces the sampling strobe pulse of optical module, and its speed is moved automatically by programming.Certainly, very clear, such sequential also can be regulated to detect and measurement signal (for example fluorescence) for the operation of light source irradiation and detector.

In another embodiment, the instrument that comprises control unit further comprises and is used for sample cell is sent into device in the hole (the slotted eye mouth of container holder that for example, comprises the heat block of liquid composition).In one embodiment, described device can be the robot system that comprises electric motor, roller bearing, anchor clamps and the necessary structure of other mobile example groove.

The specimen preparation station

Of the present invention aspect some in, device of the present invention is connected with robot sample preparation and/or sample preparation unit operation.For example, control unit can provide operation automatic sample collection, to collection tube add reagent, from described, handle and extract nucleic acid, randomly with sample transfer in new pipe, add the necessary reagent of subsequent reactions (for example PCR or order-checking) and transfer sample program to thermal cycler.In various embodiments, specimen preparation can described here Continuous Flow PCR system (Figure 11) in or in the non-continuous system (Fig. 1,5).

Carry out the method for PCR

Comprise that the thermal cycler of liquid metal or heat-conducting fluid heat block can be used for genotype identification, phylogenetic classification, environmental monitoring, paternity test and other purposes of medical diagnosis on disease, drug screening, individuality.In addition, use liquid metal or heat-conducting fluid heat block to obtain nucleic acid from any experiment source.For example, specimen can be biological sample and/or environmental sample.Biological sample can be derived from the mankind, other animals or plant, body fluid, solid tissue's sample, tissue culture or be derived from the cell of tissue culture and the offspring, contained the sample of target nucleic acid by the section of any preparation in these sources or smear or other any suspection.Typical biological sample is a body fluid, includes but not limited to blood, urine, spinal fluid, cerebrospinal fluid, knuckle synovia, amniotic fluid (ammoniac fluid), seminal fluid and saliva.The biological sample of other types can comprise food and batching, as vegetables, milk preparation, meat, meat by-product and waste material.Environmental sample is derived from surrounding material, includes but not limited to soil, water, sewage, makeup, agricultural and production piece air filter sample and air-conditioning sample.

The thermal cycler that comprises liquid metal or heat-conducting fluid heat block can be used for any scheme or experiment that needs thermal cycling or can accurately keep the heat block of uniform temperature.For example, described thermal cycler can be used for polymerase chain reaction (PCR), quantitative polyase chain reaction (q PCR), nucleic acid sequencing, ligase enzyme chain polymerization polymerase chain reaction (LCR-PCR), reverse transcription PCR reaction (RT-PCR), single base extension (SBE), multiple single base extension (MSBE), reverse transcription is connected with nucleic acid.

Instrument of the present invention allows to carry out PCR with higher speed and specificity, particularly under the situation of PCR in real time.Employing has the composition of high thermal conductivity coefficient, as liquid metal, makes it possible to carry out alternating temperature (heating up or cooling) more than the fast speed of conventional P CR.This has not only increased the potential speed of finishing PCR, and also the generation of the non-specific hybridization by reducing primer has improved the specificity of PCR.In addition, in the situation of PCR in real time, avoided sample cell is shifted out the needs that detect from heating combination from discrete parts (as the top) measurement signal of test container.This has also kept temperature control and measurement is carried out in heating cycle in real time.The reflecting material that uses anti-stop signal to overflow in addition from the discrete positions of sample cell makes it possible to use more insensitive detector, measures because can collect more light.

The PCR reaction conditions generally includes the circulation in two steps or three steps.The circulation of two steps has denaturing step, is hybridization/extension step afterwards.The circulation of three steps comprises denaturing step, is hybridization step afterwards, and the hybridization of primer and DNA chain is independent extension step subsequently in this process.The polymeric enzyme reaction thing is hybridized at primer and target sequence, and hatches under the condition by polymerase extension.The amplified reaction cycling condition is selected so that primer and target sequence are hybridized specifically and extended.

Successful pcr amplification needs high yield, highly selective and has controlled speed of reaction in each step.Output, selectivity and speed of reaction generally depend on temperature, and optimum temps depends on the composition of polynucleotide and other compositions in length, enzyme and the reaction system.In addition, the optimum temps of different step may be different.Different according to the composition of target sequence and primer, optimum reaction condition may change.Thermal cycler can be by select to need keeping temperature, each round-robin time span, cycle index, rate temperature change etc. programme.

The primer that is used for amplified reaction can be according to known algorithm design.For example, can use the algorithm of in software commercialization or customization, carrying out to design the primer of the target sequence that needs in order to amplification.Generally, the possible length of primer is at least 12 bases, more generally be 15,18 or 20 bases, but length can reach 50 more than the base.Primer is typically designed to all primers that participate in specific reaction melting temperature(Tm) difference each other in 5 ℃ scope, and more typical be in 2 ℃ of scopes.Primer is further designed to avoids primer itself or hybridization each other (priming).Primer concentration should be enough to and combine so that the target sequence amount to the accurate evaluation of the amount of extension increasing sequence to be provided through amplification.One skilled in the art will recognize that the primer concentration value will be according to the binding affinity of primer and the quantitative changeization for the treatment of the bonded sequence.Typical primer concentration scope is to 0.5 μ M from 0.01 μ M.

In one embodiment, liquid metal or heat-conducting fluid heat block can be used for the PCR reaction, and perhaps as the parts of thermal cycler, perhaps conduct is used to keep the heat block of single temperature.In typical PCR circulation, the sample that comprises DNA polynucleotide and PCR reaction mixture is by handling 10-90 second and sex change at about 90-98 ℃ in liquid metal or heat-conducting fluid heat block.Then, the polynucleotide of sex change are hybridized with Oligonucleolide primers by handling 1-2 minute under about 30-65 ℃ temperature in liquid metal or heat-conducting fluid heat block.Subsequently, by archaeal dna polymerase for carrying out chain extension with the effect of Oligonucleolide primers annealed polynucleotide.This is reflected in liquid metal or the heat-conducting fluid heat block being about under 70-75 ℃ the temperature and carried out 30 seconds to 5 minutes.According to the variable of the severity of the amount that includes but not limited to initial DNA polynucleotide, the expectation length of product and primer, can finish any PCR circulation of wanting number of times.

In another embodiment, the PCR circulation is included in 94 ℃ of about 1 minute DNA polynucleotide sex change down.The hybridization of oligonucleotide and sex change polynucleotide occurs under the temperature that is about 37-65 ℃, about one minute of time.Polymeric enzyme reaction carried out under about 72 ℃ about one minute.All reactions are to carry out in the porous plate in the container holder slotted eye that inserts liquid metal or heat-conducting fluid heat block.Carry out about 30 circulations.The scope that said temperature scope and other numerical value are not intended to limit the invention.These scopes depend on other factors, as the type of type, container or the plate of enzyme, the type of biological sample, size of sample etc.One skilled in the art will recognize that the correct easily with the need of temperature, time length and cycle index.

Reverse transcription PCR

Reverse transcription refers to by ThermoScript II (as Moloney murine leukemia virus (MMLV) transcriptase, avian myeloblastosis virus (AMV) transcriptase or its mutation) and duplicates the process that produces cDNA by mRNA, comprises using oligomerization dT primer or oligomer (as random hexamer or eight aggressiveness) at random.In PCR in real time, use usually to have the active reversed transcriptive enzyme of interior Glycosylase H (endoH).This has eliminated mRNA, makes it possible to form second chain of DNA.Usually reverse transcription was carried out as one step before PCR.In one embodiment, RT is reflected in liquid metal or the heat-conducting fluid heat block and carries out, be included in about 37 ℃ and hatched RNA sample, reversed transcriptive enzyme, necessary damping fluid and composition about one hour, under about 45 ℃, hatched about 15 minutes then, then hatch at about 95 ℃.The cDNA product is moved out of and is used as the template of PCR then.In the alternate embodiment, after the RT step followed by the PCR step, for example in a step PCR scheme.In this embodiment, all reacted constituents are present in the sample cell that is used for RT step and PCR step.But archaeal dna polymerase is closed activity and hatches and activate until the prolongation of carrying out under 95 ℃ 5-10 minute.In one embodiment, archaeal dna polymerase is owing to activity is sealed in the existence of blocking antibody, and blocking antibody is permanent deactivation in 95 ℃ incubation step.

PCR in real time

In molecular biology, real-time polymerase chain reaction is also referred to as quantitative real-time polymerase chain reaction (QRT-PCR) or dynamic aggregation polymerase chain reaction, can be used to the specific part quantitative simultaneously and dna molecular that amplification is given.It is used for determining whether particular sequence is present among the sample, and if exist, also can determine the copy number in the sample.It is the real-time form of quantitative polyase chain reaction (Q-PCR), itself is the improvement of polymerase chain reaction.

Its process is followed the general modfel of polymerase chain reaction, but DNA is carried out quantitatively the one side that Here it is its " in real time " in each round-robin amplification back.In one embodiment, DNA is undertaken quantitatively by using the fluorescence dye that inserts double-stranded DNA.In the alternate embodiment, fluorescigenic DNA oligonucleotide probe through modification is used to quantitative DNA when hybridizing with complementary DNA.

In another embodiment, real-time polymerase chain reaction combines with inverse transcription polymerase chain reaction with quantitatively low abundance messenger RNA(mRNA) (mRNA), thereby makes the researchist can be quantitatively in specified time or the relative genetic expression in specific cells or types of organization.

In specific implementations, utilize detectable marker (as the fluorescent DNA combination dye), amplified production can directly be observed.In one embodiment, amplified production is by using intercalative dye quantitative, and these dyestuffs include but not limited to that SYBR is green, SYBR is blue, DAPI, iodate third ingot, Hoeste, SYBR gold, ethidium bromide, acridine, proflavine, acridine orange, acriflavine, fluorescent coumarin (fluorcoumanin), ellipticine, daunomycin, chloroquine, distamycin D, Toyomycin, Novidium (homidium), mithramycin, ruthenium polypyridine, anthramycin.For example, dna binding dye (green as SYBR) is in conjunction with all two strandss (ds) DNA, thereby the increase of measuring fluorescence intensity, thereby can determine starting point concentration.As usual the PCR reaction mixture of preparation standard adds fluorescence ds DNA dyestuff and also joins in the sample.Be reflected at then in the liquid heat piece thermal cycler and carry out, and with photographic camera the level of fluorescence is measured in each time circulation back.In the time of on being attached to ds DNA (being the PCR product), dyestuff sends much better than fluorescence.Because the amount that is embedded into the dyestuff in the double chain DNA molecule is usually proportional with the amount of the DNA product of amplification, people are the fluorescence of the suitable quantitative intercalative dye of instrument and determine the amount of amplified production in the optical system of the application of the invention or other prior aries easily.When the use standard is diluted, can determine the concentration of PCR double center chain DNA.In some embodiments, the result who obtains for target sequence can carry out stdn at the gene (" house-keeping gene ") of stably express, as Actin muscle, GAPDH or 18srRNA.

In various embodiments, marker/dyestuff is detected by system of the present invention or device.Term " marker " or " dyestuff " refer to that can produce can be by any material of naked eyes or the detected signal of apparatus.Be applicable to that various marker of the present invention comprises the marker that produces signal by chemistry or physical means, as fluorescence dye, chromophoric group, electrochemical structure, enzyme, the radioactivity structure, the phosphorescence group, the fluorescence structure, chemoluminescence structure or quantum dot, or more particularly, radioactively labelled substance, the fluorophore marker, quantum dot-labeled thing, the chromophoric group marker, the enzyme labelling thing, the affinity ligands marker, the electromagnetism spin label, the heavy atom marker, probe with nanoparticle scattering of light mark or other nanometer particle to mark, fluorescein isothiocyanate (FITC), TRITC, rhodamine, tetramethylrhodamin, the R-phycoerythrin, Cy-3, Cy-5, Cy-7, Texas is red, Phar-is red, allophycocyanin (APC), as the Taqman probe, TaqMan Tamara probe, the probe of TaqManMGB probe or Lion probe (Biotools), as the green I of Sybr, the green II of Sybr, the Sybr gold, CellTracker is green, 7-AAD, ethidium homodimer I, ethidium homodimer II, the fluorescence dye of ethidium homodimer III or ethidium bromide, as the epitope marker of FLAG or HA epi-position, and as alkaline phosphatase, horseradish peroxidase, I ²-tilactase; alkaline phosphatase; the enzyme labelling thing of beta-galactosidase enzymes or acetylcholinesterase and as the hapten conjugates of digoxigenin or dinitrobenzene; maybe can form mixture in conjunction with right member; as streptavidin/vitamin H; avidin/biotin or immune complex (for example comprise; rabbit igg and anti-rabbit igg); as Umbelliferone; fluorescein; fluorescein isothiocyanate; rhodamine; tetramethylrhodamin; eosin; green fluorescent protein; algae is red; tonka bean camphor; methylcoumarin; pyrene; Victoria Green WPB; stilbene; fluorescent yellow; cascade indigo plant (Cascade Blue); dichloro triazinyl amine (dichlorotriazinylamine) fluorescein; dansyl chloride; phycoerythrin; the fluoresce lanthanide mixture mixture of europium and terbium (as comprise); Cy3; Cy5; the fluorophore of molecular beacon and fluorescent derivative thereof; luminophore as luminol,3-aminophthalic acid cyclic hydrazide; as the scattering of light or the plasma resonance material of gold or silver-colored particle or quantum dot, or comprise ¹⁴C, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, Tc99m, ³⁵S or ³The radioactive substance of H, or spherical shell, and the probe that produces the marker mark with any other signal well known by persons skilled in the art.For example, but detection molecules includes but not limited to fluorophore and molecule well known in the prior art, as at for example Principles of FluorescenceSpectroscopy, Joseph R.Lakowicz (editor), Plenum Pub Corp, those that describe among the sixth version Molecular ProbesHandbook of second edition (in July, 1999) and Richard P.Hoagland.

Use device of the present invention to detect intercalative dye, (for example include but not limited to phenanthridines and acridine, ethidium bromide, iodate third ingot, the own ingot of iodate (hexidium iodide), dihydro second ingot (dihydroethidium), ethidium homodimer-1 and-2, nitrine ethidium bromide (ethidium monoazide) and ACMA); Some minor groove binders (minor grovebinder) are as indoles and imidazoles (for example, Hoechst 33258, Hoechst 33342, Hoechst34580 and DAPI); And nucleic acid staining agent of all kinds, as acridine orange (also can embed), 7-AAD, dactinomycin, LDS751 and Hydroxystilbamidine.Above-mentioned all nucleic acid staining agent all can be buied from suppliers, as Molecular Probes, Inc.

In addition, the following dyestuff that is provided by Molecular Probes is provided the example of other nucleic acid staining agent: cyanine dyes, as SYTOX indigo plant, SYTOX is green, the SYTOX orange, POPO-1, POPO-3, YOYO-1, YOYO-3, TOTO-1, TOTO-3, JOJO-1, LOLO-1, BOBO-1, BOBO-3, PO-PRO-1, PO-PRO-3, BO-PRO-1, BO-PRO-3, TO-PRO-1, TO-PRO-3, TO-PRO-5, JO-PRO-1, LO-PRO-1, YO-PRP-1, YOPRO-3, PicoGreen, OliGreen, RiboGreen, the SYBR gold, the green I of SYBR, the green II of SYBR, SYBR DX, SYTO-40,-41,-42,-43,-44,-45 (indigo plants), SYTO-13,-16,-24,-21,-23,-12,-11,-20,-22,-15,-14,-25 (green), SYTO-81,-80,-82,-83,-84,-85 (oranges), SYTO-64,-17,-59,-61,-62,-60,-63 (red).Other can detect mark and comprise chemoluminescence and chromophores, optics or electron density mark etc.

As mentioned above in specific embodiment, marker comprises semiconductor nanocrystal (as quantum dot, i.e. Qdots), as describing in No. 6207392 patent of the U.S..Qdots can buy from Quantum Dot Corporation.Can be used for implementing semiconductor nanocrystal of the present invention and comprise the semi-conductive nanocrystal of II-VI family, as MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, HgTe and mixed component thereof; And the semi-conductive nanocrystal of III-V family, as gallium arsenide, InGaAsP, indium phosphide and indium arsenide and mixed component thereof.Using IV family's semi-conductor (as germanium or silicon) or using organic semiconductor also is feasible under certain condition.Semiconductor nanocrystal also can comprise two or more semi-conductive alloys that comprise selection from above-mentioned III-V compounds of group, II-VI compounds of group, IV family element and combination thereof.

Except various fluorescent DNA combination dyes, other luminous markers (as the sequence-specific probe) can be used for amplified reaction, and the detection that is beneficial to amplified production is with quantitative.Depend on the sequence-specific detection of expection amplified production based on the quantitative amplification of probe.Different with the quantivative approach based on dyestuff, its adopts luminous, the specific probe of target (for example, TaqMan

Probe), specificity and sensitivity have been increased.Carry out in this area, well being set up, and in the 5th, 210, No. 015 patent of the U.S., provide based on the method for the quantitative amplification of probe.

In another embodiment, the fluorescence oligonucleotide probe is used for quantitative DNA.It is commonly known in the art comprising the fluorescence dye that connects that base connects or terminal and the fluorescence oligonucleotide (primer or probe) of quencher.They can from, for example Life Technologies (Gaithersburg, Md.), Sigma-Genosys (The Woodlands, Tex.), GensetCorp. (La Jolla, Calif.) or Synthetic Genetics (San Diego Calif.) obtains.Base bonded fluorescence dye is modified by the synthetic back of oligonucleotide and is incorporated in the oligonucleotide, and these oligonucleotide have the reactive group that is connected with base.Those skilled in the art know, can obtain a large amount of different fluorophores, comprise from for example Molecular Probes, and Eugene, Oreg obtains, and other fluorophore is well known by persons skilled in the art.The available fluorophore comprises: fluorescein, fluorescein isothiocyanate (FITC), carboxyl Tetrachlorofluorescein (TET), the NHS-fluorescein, 5 and/or 6-carboxyl fluorescence (FAM), 5-(or 6-) iodo-acid amide fluorescein (iodoacetamidofluorescein), 5-{[2 (with 3)-5-(acetyl mercapto)-succinyl-] amino } fluorescein (SAMSA-fluorescein) and other fluorescein derivatives, rhodamine, lissamine rhodamine B SULPHURYL CHLORIDE, the texas Red SULPHURYL CHLORIDE, 5 and/or 6 carboxyl rhodamines (ROX) and other rhodamine derivatives, tonka bean camphor, 7-amino-methyl-tonka bean camphor, 7-amino-4-methylcoumarin-3-acetate (AMCA) and other coumarin derivativess, BODIPY ^TMFluorophore, as 8-methoxyl group pyrene-1,3, the Cascade Blue of 6-trisulfonic acid trisodium salt ^TMFluorophore, as 3, the fluorescent yellow fluorophore of 6-disulfonate-4-amino-naphthalimide, the phycobiliprotein derivative, Alexa fluor dyestuff (can be from Molecular Probes, Eugene, Oreg obtains) and other fluorophore well known by persons skilled in the art.For the summary tabulation of available fluorophore, also can be referring to Hermanson, G.T., BIOCONJUGATE TECHNIQUES (Academic Press, San Diego, 1996).

Use the embodiment of fluorescent tracing probe to produce accurate and reliable result.Probe based on sequence-specific RNA or DNA is used for quantitative probe sequence especially, is not at all double-stranded DNAs.This specific probe that also makes it possible to have by use the different colours mark carries out multiple analysis to several different genes in same reaction.

In one embodiment, PCR carries out in the thermal cycler that comprises the liquid metal that contains fluid composition or heat-conducting fluid heat block.In another embodiment, thermal cycler further comprises optical module.In another embodiment, liquid metal or heat-conducting fluid heat block are regulated the temperature that is included in the sample in the sample cell fast and equably and amplified production are detected in real time allowing to.In another embodiment, detection is that the marker by nonspecific nucleic acid carries out, as intercalative dye, signal index wherein, or the positive fluorescence intensity signals that produces of specific amplification products is 3 times of the fluorescence intensity that produces of PCR control sample at least, for example about 3.5,4.5,5,5.5,6,6.5,7,7.5,8,8.5,9,9.5,10,10.5 or 11 times.In one embodiment, thermal cycler can be regulated sample temperature by the speed of per second more than 10 ℃, and for example per second is 10.5 ℃.

In one embodiment, used the probe based on RNA, it has fluorescence reporter group and the quencher group that remains on close position.Fluorescence reporter group and quencher group closely near the fluorescence that has stoped itself, have only after probe is destroyed, and fluorescence just is detected.This process depends on 5 '-3 ' exonuclease activity of the polysaccharase that uses in the PCR reaction mixture.

Generally, ready reaction adds sequence-specific label probe when the beginning of reaction as usual.After the DNA sex change, label probe can be attached in the target area of template DNA with its complementary sequence on.When the PCR reactant was heated to suitable elongating temperature by liquid metal or heat-conducting fluid piece, polysaccharase was activated and DNA begins to extend.Along with the carrying out of polyreaction, its arrival is attached to the label probe place on the complementary sequence with DNA.Polysaccharase is cracked into independently Nucleotide with rna probe, and the fluorescence reporter group is separated with the quencher group.This causes the detected fluorescence of optical module to increase.Along with the carrying out of PCR reaction, increasing fluorescence reporter group discharges from its quencher group, causes fluorescence to present the increase of tangible how much levels.So just can accurately measure the initial amount of final sum of DNA.

Diagnostic use

In various application, device of the present invention can be used to in-vitro diagnosis and use, and for example, detects infective agent or pathogenic agent.In one embodiment, carry out PCR to detect various these type of materials with device of the present invention, it can be any pathogenic agent that includes but not limited to following pathogenic agent: bacterium, yeast, fungi, virus, eucaryon parasite etc.; Infective agent comprises influenza virus, parainfluenza virus, adenovirus, rhinovirus, coronavirus, first, second, third, fourth, hepatitis E virus etc., HIV, enterovirus, papillomavirus, Coxsackie virus, hsv or Epstein-Ba Er Er Shi (Epstein-Barr) virus; Bacterium comprises mycobacterium, streptococcus, salmonella, Shigella, Staphylococcus, eisseria, Rhodopseudomonas, fusobacterium or intestinal bacteria.Those skilled in the art are very clear, and PCR, order-checking and associated method are easy to be used for device of the present invention to be used for the detection of any infective agent.

An advantage of device of the present invention is the ability that can carry out ultrafast PCR, and this provides relative faster speed for diagnostic use.For some application (such as, diagnostic test in the detection of biological threat agent, the art), fast PCR is favourable.Fast an important requirement of PCR in real time is that thermal cycler allows rapid heating, cooling and heat passage, and signal generation system and follow the short cycling time of fast PCR compatible.As described herein, thermal cycler of the present invention can provide quick cooling (as, 5-17 ℃/second) and rapid heating (as, 10-44 ℃/second).For example, if desired, device of the present invention can provide the cycling time that is low to moderate 2 seconds.

In addition, this ultrafast PCR method can be by with known in amplification and produce the reagent that helps obtaining quickly the result aspect the time two that detectable signal needs and cooperate in device of the present invention and this area.These reagent are well known in the prior art, such as disclosed in No. 2005/0164219, U.S. Patent application.Wherein we one of adopting is the rapid polymerization enzyme, as KOD.

For example, special labeled primer can provide the signal of moment that (2005/0164219) takes place.The reactant generation internal rearrangement of in last circulation, extending, in a single day and reach elongating temperature, signal just produces.In the Standard PC R reaction with slow cycling condition, sort signal generation difference is also not obvious.Yet when the extension time in fast PCR shortened, this feature became an advantage and changes into fast PCR.Utilize device of the present invention, single copy bacterium sequence can be detected in less than 15 minutes.An example is to use scorpion type primer (Scorpions primer) and fast PCR instrument that low-level genus bacillus (Bacillus spp) is carried out rapid detection.In addition, depend on the amount of the DNA of input, infective agent can be detected being shorter than in time of 10 minutes, even lower level sample also can detect being shorter than in time of 14 minutes.

Embodiment

Embodiment 1

Stainless steel PCR sample cell

Stainless steel PCR sample cell is proved to be the suitable container that carries out the polymerase chain reaction therein.The container that is used for initial testing is made by stainless steel tube, and final external diameter is 0.061 inch.Long two inches sample cell is in the stainless steel plug sealing of one end with pressure fitted.

The preparation of 20 identical μ L real-time polymerase chain reactions (with the green intercalative dye of SYBR as indicator) is put into commercially available glass PCR kapillary and above-mentioned metal sample groove simultaneously.In each container, as the supernatant liquid layer reagent and extraneous air are completely cut off then by mineral oil with 20 μ L.These containers carry out in commercially available PCR in real time instrument 40 cycles of temperature cycle together, the content of metal sample groove are transferred in the commercially available glass PCR kapillary then.Two samples are set up melting curve, and the result shows that PCR completes successfully (Figure 12) in glass and metal vessel.

Embodiment 2

PCR in real time

Liquid metal or heat-conducting fluid heat block are very suitable for real-time PCR reactions, because liquid metal or heat-conducting fluid have alternating temperature speed, thermal uniformity and reflectivity fast.

By PCR is reacted composition add in the sample cell and sealed vessel to prevent to overflow or sample cell is prepared in crossed contamination.The reaction composition comprises damping fluid, target nucleic acid, suitable primer and probe, Nucleotide, polysaccharase and optional additional composition.In one embodiment, comprised four kinds of fluorescent probes, respectively be used to detect different target sequences, and specific reaction vessel can comprise any or multiple of this fluorescent probe.Each probe is advantageously reacted to the light with different incident wavelengths and is launched the light of different wave length.

Detection module is installed on the heat block.Detection module comprises four sense channels.Each access needle is optimized one in the different fluorescent probes that comprise in the sample cell.Sample cell is placed in the heat block container holder slotted eye.Cap assembly seals and is positioned on the heat block.

Each passage of detection module is calibrated, and calibration is carried out with the detection and localization module by the operation stepper-motor, makes at least one and calibrating position formation optical communication in its passage.Each calibrating position provides known fluorescence to reply.Therefore, calibration measurement can be used for correcting subsequently for the sample measurement of variable or the fluctuation of detector response.Known in the state of the art have many collimation techniques.When use had the detection module of a plurality of passages, each passage can be calibrated independently.

Carry out the PCR circulation.Thermal cycler control liquid metal or heat-conducting fluid heat block are regulating the temperature of sample cell, thereby the time that makes sample cell needing keep under needed temperature circulates with the PCR that finish two steps or three steps.Optical module scanning is also detected sample cell.Each LED channels or other light sources (as laser apparatus) are activated (glittering of short duration time) with fluorescence excitation.In one embodiment, different LED channels run parallel, and in the alternate embodiment, they move successively to avoid causing from the reflection LED light of a passage spurious response of the photodetector of another passage.The operation of optical module can be controlled by outer computer or by controller built-in in the thermal cycler.Measure with the operation of thermal cycler and carry out synchronously, can regard as corresponding to the specified time in the PCR process so that measure.In another embodiment, the function unit of thermal cycler can open and close one or more fans automatically, and these fans randomly are connected on the heat block or in conjunction with one or more scatterers.

The fluorescence that produces is detected by the corresponding light electric diode of passage or other detectors, and detector readings is sent to outer computer.This detector can read in various manners.For example, can the detection signal peak value, can add up the signal of section sometime, maybe can measure the decay that LED closes the back fluorescent signal.

These steps change with the position of each detection module and repeat, and make each passage of detection module finally detect each sample cell.In one embodiment, with the scanning of 4 passages and detect 96 sample slotted eyes each need about 15 seconds.Outside computer can be carried out a program so that the user can check the data of collecting of figure and/or tabulated form.

The real-time fluorescence measurement of above process is used for detecting and the quantitatively existence of each target sequence.This measurement also can be used for as determining speed of reaction and adjust reaction parameter raising the efficiency, and purpose that no longer need to determine the time (for example, when producing the target sequence of q.s) of extra reaction cycle in specific experiment.

Be appreciated that above process is that exemplary and multiple variation and modification are possible.The step that order is described can parallelly be carried out, and the order of step can change, and step can make amendment or make up.For example, fluorescence measurement can carry out by the arbitrary time point in the PCR working cycle, carries out repeatedly fluorescence measurement (comprising continuous sweep sample slotted eye basically) in each PCR circulation, or does not carry out fluorescence measurement after the PCR of certain number circulation.Can use the distinguishable fluorescent probe of arbitrary number in the single reaction vessel, and detection module can be provided with to comprise at least the passage with the probe similar number that uses.In some embodiments, detection module comprises a plurality of passages at identical probe optimization.This can shorten sweep time, because only need one in these passages to detect specific sample slotted eye.

Although provide and described preferred implementation of the present invention at this, those skilled in the art are very clear, and these embodiments only provide in illustrational mode.It may occur to persons skilled in the art that many do not deviate from variation of the present invention, modification, replacements.Should be appreciated that the various different alternative that when enforcement is of the present invention, can adopt embodiments of the present invention described herein.Therefore the intent of the present invention is that following claim limits scope of the present invention, and covers method and structure and Equivalent thereof in the scope of these claims.

Claims

1, a kind of method of carrying out PCR comprises:

A) carry out PCR in sample cell, and produce the signal of indication PCR process in sample cell, wherein all described signal reflexs are returned in the described sample cell basically, and wherein all described signals are detected from a discrete positions of described sample cell basically; And

B) measure from the signal of this discrete positions emission of sample cell.

2, the method for claim 1 comprises a plurality of PCR round-robin of real-time measurement signal.

3, method as claimed in claim 2, wherein said sample cell comprise the transparent wall of signal, and at least a portion of this wall immersion liquid composition, all arrive the signal of this liquid composition basically in this liquid composition reflection.

4, method as claimed in claim 3, wherein said liquid composition comprises metal or metal alloy.

5, method as claimed in claim 3, wherein said signal is produced by marker or dyestuff.

6, the method for claim 1, wherein said sample cell comprise the material of all the described signals basically in the described sample cell of reflection.

7, the method for claim 1, wherein said sample cell is made of metal.

8, the method for claim 1, wherein said sample cell comprises reflecting material.

9, method as claimed in claim 2, wherein said sample cell is isolated by container holder and described liquid composition.

10, a kind of method of carrying out PCR comprises:

A) under the following conditions the target nucleotide sequences in the reaction mixture is carried out the PCR reaction:

I) between primer extension and double-stranded the dissociating and the rate temperature change between primer annealing and the primer extension surpass 10.5 ℃ of per seconds;

Ii) described PCR carries out in comprising the heat block of sample cell, and temperature contrast is less than 0.5 ℃ in the sample cell; And

B) monitor the amplification of target nucleotide sequences in real time.

11, method as claimed in claim 10, wherein said heat block comprises liquid composition.

12, method as claimed in claim 10 further is included in the temperature contrast measured between two or more holes of described heat block less than 0.5 ℃.

13, method as claimed in claim 12, wherein the described temperature contrast of measuring between two or more holes of described heat block is 0.01 ℃.

14, method as claimed in claim 10, wherein said heat block are swap block.

15, method as claimed in claim 10, wherein variation of temperature speed realizes in the following manner:

(1) sample cell that will comprise reaction mixture with have the liquid composition thermo-contact of the heat transfer coefficient of 0.1W/MK at least; Wherein the temperature of liquid composition is controlled the temperature of reaction mixture.

16, method as claimed in claim 15 comprises that the motion by luring liquid composition into keeps temperature homogeneity.

17, a kind of method of carrying out PCR comprises: carry out the PCR reaction in the thermal cycler with the speed setting sample temperature that is higher than 5 ℃ of per seconds; The temperature of wherein said thermal cycler is regulated by the liquid composition in the heat block of liquid sealing; The heat transfer coefficient of wherein said liquid composition is at least 0.1W/MK.

18, method as claimed in claim 17, wherein said thermal cycler provide the alternating temperature speed at least about 10 ℃ of per seconds.

19, method as claimed in claim 17, wherein said thermal cycler provide at least about between 0.01 ℃ to 0.1 ℃ Kong Yukong and temperature homogeneity sample.

20, method as claimed in claim 18, wherein said heat block have 0.01 ℃ the temperature contrast of measuring between two or more holes of described heat block.

21, method as claimed in claim 18, wherein said liquid composition is contained in the heat block.

22, method as claimed in claim 20, wherein said heat block are swap block.

23, a kind of method of carrying out PCR comprises: regulating sample temperature fully so that carry out PCR to be at least in the thermal cycler that 3 signal index detects in real time to amplification; Wherein said detection realizes that by non-specific nucleic acid marking wherein said thermal cycler is with the speed setting sample temperature of per second more than 10 ℃.

24, a kind of method of carrying out PCR in real time comprises:

A) in reaction mixture between primer extension and double-stranded the dissociating and under the condition of the rate temperature change between primer annealing and the primer extension for 15 ℃ of per seconds at least, in reaction mixture, carry out the PCR reaction of target nucleotide sequences, wherein said PCR be reflected at the situation with liquid composition generation thermo-contact of the heat transfer coefficient of 0.1W/mK at least under carry out; And

B) PCR is monitored in real time.

25, method as claimed in claim 24, wherein monitoring comprises:

A) carry out PCR in sample cell, and produce the signal of indication PCR process in sample cell, wherein signal is launched from sample cell from a discrete positions basically; And

B) measure from the signal of this discrete positions emission of sample cell.

26, method as claimed in claim 24, wherein the heat-up rate in the reaction mixture is 40 ℃ of per seconds at least.

27, method as claimed in claim 24, wherein said temperature variation is regulated by Peltier's element.

28, a kind of method of carrying out PCR in real time comprises:

A) temperature of PCR reaction mixture dissociated in two strands, carry out a plurality of circulations between the temperature of primer annealing and primer extension, wherein each circulation was no more than for 5 seconds; Wherein said temperature is regulated by the liquid composition that is enclosed in the thermal cycler; And

B) the PCR process in the real-time monitoring sample cell in a plurality of circulations.

29, method as claimed in claim 28 also comprised before step a):

1) make the blind end of the sample cell that comprises the PCR reaction mixture and liquid composition form thermo-contact, this liquid composition is being liquid more than 60 ℃ and is having the heat transfer coefficient that is at least 0.1W/mK, thus the temperature of the temperature of liquid composition control PCR reaction mixture.

30, method as claimed in claim 28, wherein all basically light in the liquid composition reflection sample cell.

31, method as claimed in claim 30, wherein monitoring is to be undertaken by measuring from the signal of sample cell top or bottom emission.

32, method as claimed in claim 28, wherein each circulation is no more than 3 seconds.

33, method as claimed in claim 29, wherein said sample cell comprises reflecting surface.

34, method as claimed in claim 29, wherein said sample cell further covers with lid.

35, method as claimed in claim 34, wherein said lid can perhaps be cooled off by described liquid composition from described liquid composition heat absorption.

36, a kind of method of carrying out PCR in real time comprises thermal cycler is provided that this thermal cycler comprises the liquid composition with PCR sample cell generation thermo-contact; Wherein said liquid composition carries out temperature regulation, thus the temperature of reaction in the regulation and control sample cell, the wherein detectable signal of emission from described sample cell; And detect described signal by the optical module that comprises optical transmitting set and fluorescence detector.

37, method as claimed in claim 36, wherein said optical module comprise pin photodiode, CCD imager, cmos imaging instrument, line scanner, photorectifier, phototransistor, photomultiplier cell or avalanche photodide.

38, method as claimed in claim 36, wherein said sample cell further covers with lid.

39, method as claimed in claim 38, wherein said lid can perhaps be cooled off by described liquid composition from described liquid composition heat absorption.

40, a kind of instrument comprises:

A) temperature-controlling module, comprise the container that comprises liquid composition, described liquid composition is being liquid more than 60 ℃ and is having the heat transfer coefficient of 0.1W/mK at least, described container has at least one hole, each hole is suitable for holding the blind end of sample cell, the sample cell and the liquid composition that wherein are contained in the hole form thermo-contact, thus the temperature of liquid sample in the temperature of the liquid composition control sample cell, and wherein liquid composition can reflect light all basically in the sample cell;

B) optical module can detect the light in the described sample cell from a discrete positions on the described sample cell; And

C) control unit, the temperature of its controlled liq composition and the operation of optical module.

41, instrument as claimed in claim 40, wherein liquid composition comprises gallium, gallium-indium alloy or comprises gallium, indium, rhodium, silver, zinc, tin or stannous alloy.

42, instrument as claimed in claim 40, wherein temperature regulator comprises Peltier's element.

43, instrument as claimed in claim 40, wherein temperature regulator comprises the resistance wire with the liquid composition thermo-contact.

44, instrument as claimed in claim 40, wherein temperature regulator comprise that the temperature that makes liquid composition is dissociated in the two strands that is suitable for PCR, round-robin device between the temperature of primer annealing and primer extension.

45, instrument as claimed in claim 40, wherein thermal cycler also comprises the device that the liquid stream that makes in the liquid composition circulates.

46, instrument as claimed in claim 40, wherein said optical module comprises optical transmitting set and fluorescence detector.

47, instrument as claimed in claim 40 further comprises:

Comprise the specimen preparation station of in sample cell, adding the device of reagent.

48, instrument as claimed in claim 40 further comprises sample cell is moved to device in the hole.

49, instrument as claimed in claim 47 further comprises the digital machine of controlling thermal cycler, optical module and specimen preparation station.

50, a kind of system that carries out PCR in real time comprises:

A) thermal cycler comprises liquid composition and is used to engage sample cell and makes described sample cell and the device of liquid composition formation thermo-contact; And

B) optical module comprises optical transmitting set and fluorescence detector, and it imports light into the sample cell of joint and detects from the light of the sample cell emission that engages.

51, system as claimed in claim 50, wherein said liquid composition comprises metal or metal alloy.

52, system as claimed in claim 50, wherein said liquid composition comprises gallium.

53, system as claimed in claim 50, wherein said transmitting from the removable lid of described sample cell.

54, system as claimed in claim 50, wherein said liquid composition is isolated by container holder and described sample cell.

55, system as claimed in claim 54, wherein said container holder is transparent or translucent.

56, system as claimed in claim 50, wherein said system comprises Peltier's element.

57, system as claimed in claim 56, wherein said thermal cycler also comprises the electric motor that is operatively connected with fan and stirring rod.

58, system as claimed in claim 57, wherein said fan is connected with described electric motor is coaxial with stirring rod.

59, system as claimed in claim 50, wherein said thermal cycler also comprises the resistance wire of the heat regulation and control that are used for described liquid composition.

60, system as claimed in claim 50, wherein said liquid composition is sealed in the confinement barrier, and wherein said barrier comprises the surface with container holder, and described sample cell places this container holder.

61, system as claimed in claim 50, wherein said liquid composition directly contacts with described sample cell.

62, system as claimed in claim 50, wherein said optical module comprises pin photodiode, CCD imager, cmos imaging instrument, line scanner, photorectifier, phototransistor, photomultiplier cell or avalanche photodide.

63, a kind of instrument comprises,

A) scatterer;

B) heating unit that contacts with described radiator heat;

C) comprise the barrier of the wall with end face and bottom surface, wherein the bottom surface is sealed on the described heating unit, and wherein Mi Feng barrier and described heating unit form the container that comprises liquid composition;

D) comprise first lamellar body of a plurality of slotted eyes, wherein this first lamellar body is sealed on the end face of barrier and slotted eye extends in the container;

E) comprise second lamellar body of a plurality of sample cells, each sample cell has opening end, and wherein sample cell inserts in the slotted eye removedly;

F) comprise the 3rd lamellar body of a plurality of projections, wherein projection is inserted the opening end of sample cell removedly.

64, as the described instrument of claim 63, it is liquid and the metal with the heat transfer coefficient that is at least 0.1M/W/K that wherein said liquid composition is included in more than 60 ℃.

65, as the described instrument of claim 63, the volume of wherein said liquid metal can increase about 0.1 to 6.0%.

66, as the described instrument of claim 63, wherein said intensification ability surpasses 10.5 ℃ of per seconds.

67, as the described instrument of claim 63, wherein e) described in slotted eye be set to Yi Shixing so that described slotted eye energy and f) described in sample cell form heat or optics contacts.

68, as the described instrument of claim 67, wherein said setting is included in and adopts geometric configuration and/or deformable material in the described slotted eye.

69, as the described instrument of claim 68, wherein said geometric configuration is Polygons, ellipse or circular.

70, as the described instrument of claim 63, wherein said slotted eye immerses described liquid composition to initial level.

71, as the described instrument of claim 70, wherein said projection extends to that described initial level is above, described initial level place or below the described initial level.

72, as the described instrument of claim 63, wherein said the 3rd lamellar body can cool off from described liquid composition absorption heat or by described liquid composition.

73, as the described instrument of claim 63, the slotted eye of wherein said first lamellar body is transparent and Yi Shixing, and the sample cell of described second lamellar body is transparent, learns and contacts thereby the enhanced pressure on described slotted eye makes sample in described liquid and the described sample cell in the described container form light and heat.

74, as the described instrument of claim 63, the projection of wherein said the 3rd lamellar body is transparent.

75, as the described instrument of claim 63, wherein said barrier by gasket seal on the radiator and first lamellar body.

76, as the described instrument of claim 63, wherein said first lamellar body or second lamellar body comprise reflecting surface.

77, as the described instrument of claim 63, wherein said barrier utilizes fastener to be sealed on the radiator and first lamellar body.

78, as the described instrument of claim 63, also comprise the radiator that contacts with described heating unit.

79, as the described instrument of claim 63, wherein said heating unit is Peltier's element, kind of thread elements or its combination.

80, as the described instrument of claim 63, also comprise second heating unit, wherein said barrier is placed between described heating unit and described second heating unit.

81, as claim 63 or the described instrument of claim 69, wherein said a plurality of slotted eyes comprise 4,8,16,32,48,96,196,384 or 1536 slotted eyes.

82, as claim 63 or 69 described instruments, also comprise fan and stirring rod, wherein randomly described fan and stirring rod and monomotor are operatively connected.

83, as the described instrument of claim 82, wherein said instrument provides power by battery.

84, as the described instrument of claim 82, wherein said fan is opened and closed automatically by the function unit that is connected with described instrumentation.

85, as the described instrument of claim 63, also comprise the heat dissipation metal body.

86, as the described instrument of claim 85, wherein said radiator comprises copper.

87, as the described instrument of claim 63, wherein said disposition of heating component is for being used for grads PCR.

88, a kind of Continuous Flow PCR system comprises the specimen preparation module; Thermal cycler, wherein said thermal cycler comprises the liquid composition in order to the temperature of regulating described sample; And be used to detect the optical module that transmits from described sample.

89,, also comprise sampling thief and waste collection device as the described system of claim 88.