WO2014127806A1

WO2014127806A1 - Dna amplifying kit

Info

Publication number: WO2014127806A1
Application number: PCT/EP2013/053288
Authority: WO
Inventors: Yasuaki Kuwata; Masahiro Fujimoto; Kouichi Tsuruta
Original assignee: Daiken Medical Co., Ltd.; Mewburn Ellis Llp
Priority date: 2013-02-19
Filing date: 2013-02-19
Publication date: 2014-08-28

Abstract

The present invention is a DNA amplifying kit for amplifying a specific target DNA(s) in parallel which exists in a sample containing DNA, comprising: a sample collecting means for collecting part of a sample containing DNA; a DNA extraction reagent for extracting DNA from the collected sample to obtain DNA extract; a PCR reagent for amplification reaction of DNA on the DNA extract, comprising thermostable DNA polymerase, buffer, deoxynucleoside triphosphates, and labeling agent being spectroscopically detectable by reacting with the amplified DNA; a plural types of primer reagents each comprising one or more kinds of primers for amplification of target DNAs, each primer corresponding to each of the target DNAs; a DNA extraction container for providing a field for DNA extraction reaction by use of the DNA extraction reagent; an extract collecting means for collecting part of DNA extract obtained in the DNA extraction container; and a substrate containing at least the same number of micro flow channels as the number of the target DNAs or of groups thereof, in the micro flow channels the DNA extract collected by the extract collecting means, the PCR reagent and the primer reagent being loaded, and a field for DNA amplification reaction being provided, wherein primer(s) is allowed to be introduced in each of said micro flow channels; and wherein one primer is not introduced in two or more micro flow channels.

Description

DNA AMPLIFYING KIT

TECHNICAL FIELD

[0001]

The present invention is related to a DNA amplifying kit .

More specifically, the invention is related to a DNA amplifying kit for amplifying a spec i f i c target DNA ( s ) in parallel which exists in a sample containing DNA, and is suitably employed for a diagnosis for infection.

RELATED ART

[0002]

For treating the infection, it is general ly necessary to identify the pathogen ( s ) involved in developing the infection, and then to admini ster drugs such a s antibiotics for wiping out the pathogen ( s ) . For identifying the pathogen ( s ) , traditional culture method is idely empl oyed . Such culture method requires a high level of technical knowledge and skill ( JP 2011-512159 A : Patent Document 1) .

[0003]

Further , JP 2001-204470 A (Patent Document 2 ) proposes a method for researching if a microorganism is included in a sample collected from a test body without going through the culture method . Patent Document 2 discloses a method including conduct ing DNA ampl i fication in accordance with PGR (polymerase chain reaction) method, staining the amplified DNA with fluorescence agent such a s ethidium bromide , detecting DNA amplification by agarose electrophoresis, and thereby identifying a target DNA of the pathogen which causes infection by the following method : preparing a DNA chip on which a probe being specifically bound to the target DNA is immobi 1 i z ed ; and bring the sample solution in which the DNA ampl i fication is detected to contact with the DNA chip to hybr idi z e the amplified DNA with the probe , then identifying whi ch probe involves the hybridization .

[0004]

WO 2009/110473 ( Patent Document 3 ) discloses a technique for identifying pathogen ( s ) involved in developing infection by the following method : extracting DNA from a sample collected from a test body , amplifying the DNA, detecting if target DNA ( s ) of the pathogen ( s ) exists in the sample by use of probe ( s ) being spec ifically bound to the target DNA, quant ifying the target DNA which is detected, and then comparing the developing level a s predetermined.

SUMMARY OF THE INVENTION

[0005]

I n the culture method of Patent Document 1 , the cultural operation of the sample collected from the test body is conducted with the prediction of the pathogen ( s ) from the symptoms. Therefore, the cultivation for the pathogen with the low level of the occurrence frequency tends to be laid over. Further , it is difficult to cultivate a pathogen on which the cultivation method is not e s t abl i shed . Moreover , there is possible a pathogen whi ch may not be ident i f ied because of its biochemical characters , even if the cultivation is successful . Owing to the above circumstances, it is difficult to practice the appropriate treatment for the infection over the patient , since the immediate di agno s i s is failed to be made .

[0006]

I n the culture method, a microorganism is f i shed out of colonies appearing by cultivation, and then the f i shed microorganism is ident i f ied . Since the condition for identifying is di f fer ent from each of pathogens , di agno s i s is complicated . I n particular, for community-acquired pneumonia , the immediate di agno s i s is difficult . Moreover , for the infection caused by a plural of pathogens , it requires some amount of time to identify all of the pathogens . I n view of these circumstances, it is di f f i cul t to practice the

appropriate treatment for the infection over the patient .

[0007]

I n the techniques of Patent Document s 2 and 3 , it is necessary to take operations for identifying pathogen ( s ) i nvolved in deve loping infection separately, after the ampl i fication of DNA . I n view of reali z ing the immediate and simple diagnosis, those techniques are still insufficient.

[0008]

The present invention is accomplished in order to solve the above mentioned problems, and has intent to provide an immedi at e and simple di agno s i s technique for identifying pathogen ( s ) involved in developing infection, wherein there are a plural of pathogens which may be i nvo 1ved .

[0009]

Namely, the present invention is directed to the following :

(1) A DNA amplifying kit for amplifying a specific target DNA ( s ) in parallel which exists in a sample containing DNA, compr i sing:

A sample collecting means for collecting part of a sample containing DNA ;

A DNA extraction reagent for extracting DNA from the collected sample to obtain DNA extract ;

A PGR reagent for ampl i fication reaction of DNA on the DNA extract , compr i s i ng thermostable DNA polymerase , buffer , deoxynucleoside triphosphates, and labeling agent being spectroscopically detectable by reacting with the ampl i f i ed DNA;

A plural types of primer reagent s each compr i sing one or more kinds of primers for amplification of target DNAs , each primer corresponding to each of the target DNAs ; A DNA extraction container for providing a field for DNA extraction reaction by use of the DNA extraction reagent;

An extract collecting means for collecting part of DNA extract obtained in the DNA extraction container; and

A substrate containing at least the same number of mi cro flow channels as the number of the target DNAs or of groups thereof , in the mi cro flow channels the DNA extract collected by the extract collecting means , the PGR reagent and the primer reagent being loaded, and a field for DNA ampl i fication reaction being provided,

Wherein primer ( s ) is allowed to be introduced in each of the mi cro f low channels; and

Wherein one primer is not introduced in two or more micro flow channel s .

(2) The kit a s specif i ed in (1), wherein said sample collecting means is made up of an element f ormed in a manner to hold said sample and remove the moisture of the sample , and an element for collecting the dehydrated sample .

(3) The kit as specif i ed in any of (1) or (2), wherein said substrate further compri ses a micro f low channel for a positive control .

(4) The kit as specif i ed in (3) , wherein a template DNA having the different sequence from any of the target DNAs and a primer having the sequence complementary to a part of the sequence of said template DNA are introduced in advance to said micro flow channel of the positive control.

(5) The kit as specif i ed in (3) or (4) , wherein said substrate further comprises a micro flow channel for a negative control.

(6) The kit a s specified in any of (1) to (5), wherein the substrate is provided with a feed for introducing the DNA extract; and wherein each of micro flow channels is configured in a manner that one end of each channels connects with the feed of said substrate and the other end of each channels is open, respectively.

(7) The kit as specified in (6) , further compr i sing a sealing means for sealing the open end of each of the micro flow channel s .

(8) The kit as specified in ( 7 ) , wherein said sealing means is mineral oil.

(9) The kit as specif i ed in any of (1) to (8), wherein said primer reagent ( s ) is pre 1 imi nary introduced in said mi cr o flow channel ( s ) of said substrate .

(10) The kit a s specified in any of (1) to (9), further comprising an instruction for guiding an operator in operating said kit .

[0010]

By virtue of the present invention, it becomes possible to identify pathogen ( s ) involved in causing infection immediately and easily. As a consequence, the appropriate treatment for the infection should be effectively practiced, so that it is possible to avoid the spread of the infection.

BRIEF DESCRIPTION OF DRAWINGS

[0011]

FIG. 1 shows a diagram illustrating an embodiment of the substrate employed for the embodiment of the invention ;

FIG. 2 shows a diagram illustrating another embodiment of the substrate employed for the embodiment of the i nvent i on ;

FIG. 3 shows a diagram illustrating an embodiment of a substrate holder empl oyed for the embodiment of the invention ;

FIG. 4 shows a cross-section view of a concrete embodiment for the substrate holder of FIG. 3 ;

FIG. 5 shows a cross-section view of an alternate for the substrate holder of FIG. 3 ;

FIG. 6 shows a diagram illustrating a function of means for sealing empl oyed for the embodiment of the invention ;

FIG. 7 shows a t emperature profile in controlling temperature for the DNA amplification in the embodiment of the i nvent ion;

FIG. 8 shows a diagram illustrating a thermal cycler in the embodiment of the invention ;

FIG. 9 shows a principal diagram illustrating the thermal cycler of FIG. 8 ; and

FIG. 10 shows a f lowchar t illustrating the embodiment of the invention .

DETAILED DESCRIPTION [0012]

The invention is described be 1 ow .

From one aspect, the present invention provides a pathogen diagnosis system for identifying pathogen (s) involved in developing infection wherein there are a plural of pathogens which may be involved.

The pathogen diagno s i s system compr i s e s a body fluid collecting means for collecting part of body fluid be i ng provided from a test body and containing DNA ; a DNA extraction reagent for extracting DNA from the collected body fluid by the body fluid collecting means to obtain DNA extract ; a PGR reagent for amplification reaction of DNA on the DNA extract , compr i sing thermostable DNA polymerase , buf f er ,

deoxynucleoside triphosphates, and labeling agent being spectroscopically detectable by reacting with the ampl i f i ed DNA ; a plural types of primer reagents each compr i sing one of more kinds of primers for ampl i fication of a target DNA having sequence distinguishing each of a plural of pathogens , each primer corresponding to each of the pathogens ; a DNA extraction container for providing a field for DNA extraction reaction by use of the DNA extraction reagent ; an extract collecting means for collecting part of DNA extract obtained in the DNA extraction container ; a substrate containing at least the s ame number of mi cro f 1 ow channel s a s the number of pathogens or of groups of pathogens to be detected, in the micro f 1 ow channels the DNA extract collected by the extract collecting means, the PGR reagent and the primer reagent being loaded, and a field for DNA amplification reaction being given; and a thermal cycler for controlling the temperature for DNA amplification reaction in the mi cr o flow channel by heat i ng or cooling the substrate .

[0013]

A body fluid collecting means is a piece of equipment for collecting body fluid from a patient who is a test body, and includes a cotton-tipped applicator , a dropper , a pipette and the like. Among these, a cotton-tipped applicator is suitable in view of easy-to-use of handl ing . When it is di f f i cul t to directly collect a sample , collection may be conducted first by taking up the body fluid by use of something to hold the body fluid, such a s petr i dish and filter paper , and then by collecting a sample therefrom by use of a cot ton-tipped appl i cat or , a dropper , a pipette and the like. Further , in holding the body fluid, it is preferable to remove the moi sture of the fluid, since it becomes easier to take up a sample . Spec ifically, a filter paper is employed a s an element to take up a body fluid, and then folded with placing the body fluid i nward so that the moi sture is allowed to be absorbed, and then unfolded and sub ected to sampling by use of an element to collect the dehydrated body fluid, such a s a cotton-tipped appl i cat or , a dropper , a pipette and the like. Such a combination of those elements in order to realize the collection of body fluid is also included by the scope of the body fluid collecting means.

[0014]

For the embodiment of the invention, each of r eagent s , such as a DNA extraction reagent for extracting DNA from the body fluid, and a PGR reagent for amplification reaction of DNA, and primer reagents.

[0015]

The DNA extraction reagent contains enzyme ( s ) for destructuring the cell walls of the cell contained in the body fluid being collected from the test body , and enzyme ( s ) for breaking up proteins contained in the body fluid, and en z yme ( s ) for breaking up mucus contained in the body fluid, and also contains a component for changing a form of DNA existing in the cell so that DNA is allowed to be easily extracted, such a s a surfactant including SDS ( sodium dodecy 1 sulfate ) . Further , the commercially available DNA extraction reagent may be suitably employed, in particular , two -component type to allow the reaction to proceed in two stages is preferable , in view of handl i ng .

[0016]

The PGR reagent contains thermostable DNA polymerase, buf f er , deoxynucleoside triphosphates (dNTP), and labeling agent be i ng spectroscopicaily detectable by reacting with the amplified DNA. The thermostable DNA polymerase includes a DNA polymerase suitable for ordinary PGR reaction, such as a DNA polymerase derived from thermus aquat i cu s ( US Patents

4,889,818 and 5,079,352) (Trade name: Taq Polymerase) , a DNA polymerase derived from Thermus thermophilus (WO 91/09950) ( rTth DNA polymerase ) , a DNA polymerase derived from Pyrococcus f ur iosus (WO 92/9689) (Pfu DNA polymerase ; manufactured by Stratagenes Corp . ) , a DNA polymerase derived from Thermococcus litoralis ( EP-A^™ 455430 ) (Trade mark : Vent ; New England Biolabs Inc. ) . Further , for example , a polymerase being def i ci ent in 5 ' to 3 ' exonuclease activity is preferable . By using such a polymerase being deficient in 5 ' to 3 ' exonuclease activity, it could be avoided to cleave a primer by a polymerase . Such a polymerase being de f i ci ent in 5 ' to 3 ' exonuclease activity as commercially avail able includes for example Gene Taq (NIPPON GENE CO . , LTD . ) , TITANIUM Taq ( Clontech Laboratories Inc . ) , ATth ( TOYOBO CO . , LTD . ) .

[0017]

Moreover , buf f er s and dNTPs include ordinary product s for PGR reaction . For example, dNTPs include dATP, dCTP, dGTP, dTTP and dUTP , and the mixture thereof is suitably employed . For example, buf f er s include a solution exhibiting a suitable pH for the DNA ampl i fication by PGR method, such a s Tr i s-HCl , Tricine, MES , MOPS , HEPES and CAPS . Furthermore , labeling agent includes a compound being capable of labeling DNA by reacting with DNA . Typical examples include fluorescence agent emitting fluorescence by binding to the amplified DNA, such a s SYBR(tm) GREEN and ethidium bromide .

[0018]

Further , the PGR reagent may contain solvent such as water and buf f er , glycerol, heparin, betaine, KC1, MgC 1₂ and MgS0₄.

[0019]

The primer reagent contains one or more kinds of primers, each of which is employed for amplifying target DNAs, respectively, each having a distinctive sequence for the corresponding pathogens to be detected. That is, in the case that the pathogen(s) to be detected exist in the body fluid from the test body , the primer ( s ) is to initiate the DNA amplification reaction by PGR method by use of the extracted DNA ( s ) derived from pathogen ( s ) a s a template . Since the target for the diagnosis is the infection wherein there are a plural of pathogens which may be involved, the primer reagents should be separately prepared for every primer corresponding to each of these pathogens. Alternatively, the primer reagent may be a group of primers , each primer be i ng employed for the ampl i fication of DNAs , correspond to a plural of pathogens which does not influence on one another. It should be noted that each of group s contains the different primers from one another .

[0020] A DNA extraction container has a certain amount of volume, and provides a field for DNA extraction reaction of the body fluid collected by the body fluid collecting means by use of the DNA extraction reagent. The DNA extraction container is suitable in a form of a plastic tube , for example a mi cr o tube , in view of handling . The tube is preferred to be siliconized on the inner surface .

[0021]

11 is po s s ible to conduct the extraction reaction in the DNA extraction container . However , it is preferable to appropriately regulate the temperature of the reaction in view of reaction efficiency.

For example, the following temperature regulation may be effective : the reaction mixture is first heated to around 70 degrees C, to thereby activate en z yme s included in the DNA extraction reagent , and then the reaction is initiated ; and the reaction mixture is allowed to keep that condition for 6 minutes or so, to thereby progress the extraction reaction; then , the temperature of the reaction mixture is e 1 evat ed to 94 degrees C, to thereby inactivate the enzymes and to terminate the reaction .

Such a temperature regulation may be easily realized by use of a temperature regulator for regulating the temperature for initiating or stopping the extraction reaction in the DNA extraction container , such a s heating blocks . [ 0 0 2 2 ]

The PGR reagent may be introduced into the extraction reaction resultant by the DNA extraction reaction (hereinafter referred to " DNA extract") . Howe er , it is preferable to introduce the PGR reagent into the DNA extraction container together with the DNA extraction reagent in view of workability, since the activating temperature of the enzymes of the DNA extraction reagent is different from that of the PGR reagent.

[ 0 0 2 3 ]

An extract collecting means is a piece of equipment for collecting part of the DNA extract in the DNA extraction container, including for example a dropper and a pipette in the case of collecting as an extract the supernatant of the solution, and a tube with a membrane filter in the case of removing the other substances than the extract . When empl oyi ng the tube , it may be combined with the DNA extraction container .

[ 0 0 2 4 ]

A substrate contains a feed for introducing the DNA extract ; and a plural of micro f low channels, each being conf igur ed in a manner that one end is connected with the feed for introducing the DNA extract , and that the other end is open .

Further , the substrate is made from a material whi ch is unr eact ive with any of the DNA extraction reagent , the PGR reagent nor the primer reagent , preferably quart z in view of its excellent thermal conduct ivity . Moreover , there is no limit in the configuration .

[0025]

One of concrete embodiments is described, for example, in W02012/ 001972. That IS, 3. S shown in FIG. 1 , the substrate 40 is made up in a circle form and has a hole at the center which becomes a feed 11 for the DNA extract . Further , eight of mi cr o f low channel s 21a, 21b, 21c, 2 Id, 21 e , 21 f , 21 g and 2 Ih are formed in the s ame length to radiate from the center part of the substrate 40 , in each of whi ch one end is connected with the feed 11 , and the other end, that is, the end at side of the outer circumference of the substrate 40 , is open .

[ 0026 ]

Further , the substrate 40 has the discoid shape with about 10 mm in diameter and about 0.3 mm in thickness . The cross-section surface of the micro f low channels is 100 μιη s quare in size. By conf igur ing the substrate and the mi cro f low channel s in such form and size, it is r eal i z ed to cut down the volume of the test solution to some microliters in total for all micro f 1 ow channels, so that heat content required for the test solution can be significantly reduced .

[0027]

For the procedure in loading the pr imer s in the substrate 40 , the procedure is conducted for each of micro flow channels 21a to 21 h , separately : first, a primer reagent is dropped at the opposite end of the feed 11 by use of a dropper and the like and introduced into the channel by the capillarity effect, so that the different primer reagent is introduced into each of the mi cr o flow channels, respectively . Thereafter, the substrate is subjected to drying, so that a primer is loaded in each of mi cro flow channel s .

[0028]

A DNA extract is introduced into the micro flow channels from the feed 11 by the capillarity effect . That is, when the DNA extract is brought into contact with the feed 11 , the extract acti ely f lows in the direction indicated by arrows of FIG. 1 in each of the micro f low channel s 21a, 21b, 21c, 2 Id, 21 e , 21 f , 21 g and 21 h by the capillarity effect, so that each of the mi cro f low channel s 21a, 21b, 21c, 21 d , 21 e , 21 f , 21 g and 2 Ih will be filled surely with the DNA extract by its surface tension effected at the open end of each of the mi cro f low channel s .

[0029]

I n FIG. 1 , it is illustrated that the substrate is conf igured in a circle form . However, a s shown in FIG. 2 , the substrate may be conf igured in a rectangular or square form .

I n FIG. 2 , from planer view of the substrate 41, the substrate 41 has the mi cro f 1 ow channels 22a to 22s and 23a to 23s which are formed in parallel to one another in the direction of one s ide and arranged in the direction of the other side at certain interval , such a s 0.5 mm . I n the substrate 41 , at the side of one end of each of micro flow channels a feed lib for the DNA extract , which is conf igured in a window form in the direction of lining up the micro f low channel s in planar view of the substrate 41. Further , on the other side of the micro f low channel s , a series of primer feeds 11a is formed for introducing the primer reagents. The number of the micro flow channels is at least the same number of pathogens to be detected or groups of pathogens to be detected.

[0030]

Further , for the procedure in loading the primers in the substrate 41, the procedure is conducted for each of micro flow channel s 22a to 22s, separately : first, a primer reagent is dropped at one of the primer feed 11a by use of a dropper and the like and introduced into the channel by the capillarity effect, so that the different primer reagent is introduced into each of the micro flow channels, respectively . Thereafter, the substrate is sub ected to drying, so that a primer is loaded in each of mi cro f low channel s . A DNA extract is introduced into the mi cro flow channels from the feed lib by the capillarity effect . I n FIG. 2 , although there is not a window like the primer feeds 11a at the opposite side of the feed lib of each of the mi cro flow channels 23a to 23s, those micro f 1 ow channels 23a to 23s may be used as negat i ve control (s) a s mentioned below . I f the micro flow channels 23a to 23s are also to be used a s a space for detecting the DNA ampl i fication, a window like the primer feed 11a may be configured at the opposite side of the feed lib of each of the micro flow channels 23a to 23s.

[0031]

Prior to the DNA amplification, a primer is introduced in each of micro flow channel s in the substrate . 11 is needed to make sure that one kind of primers is never introduced in two or more micro flow channel s ; that is, each of the mi cro flow channel s contains the different primer ( s ) from one another I n particular, when one kind of a primer is in one mi cro flow channel , it means that one sole different primer is introduced in each of channels. Even when a group of a plural of primers is in one mi cro flow channel, it is needed to make sure that one kind of primers is never included in two or more groups , so that the s ame primer never exists in two or more mi cro flow channel s .

[ 0032 ]

According to such a configuration, in the micro flow channels, the PGR reagent and the primer (s) are loaded in advance ; once the extraction reaction resultant be i ng collected by the extract collecting means is introduced, a field for the DNA amplification reaction is provided under the prescribed condition of heating by the temperature control for the DNA amplification reaction as mentioned below.

[ 0033 ]

Further , it is preferable to arrange a mi cro f low channel for a positive control.

Here, the term "a positive control" means a control for representing a state that DNA amplification reaction takes place , so that the criteria for evaluating whether DNA ampl i fication reaction takes place or not in a micro f 1 ow channel , whi ch is treated for DNA ampl i fication reaction of the target DNA, may be provided . That is, by comparing the micro f 1 ow channel of the target DNA with the positive control in the detection level ( as mentioned be 1 ow ) , it may be evaluated whether the DNA amplification reaction takes place or not in the mi cro f low channel of the target DNA .

Therefore, to the micro f low channel of the positive control are introduced in advance a template DNA (hereinafter referred to "standard t emp late") having the different sequence from any of the target DNAs and a primer ( hereinafter referred to " standard primer" ) having the sequence compl ement ary to a part of the sequence of the standard template . Her e , the term "the detection level" means a spectroscopic intensity of the 1 abe 1 i ng agent ; the higher detection level shows "positive", which means that the labeling agent is detected in a higher amount : and the lower detection level shows "negative " , which means that the labeling agent is detected in a smaller amount .

According to such a configuration, to the micro f low channel of the positive control is introduced the DNA extract and the temperature control as mentioned be 1 ow is conducted, so that the DNA amplification reaction takes place between the standard primer and the standard template. In other words, if a DNA amplification reaction does not take place in the positive control , it may be evaluated that the reaction condition such a s a temperature cycle is not appropriate .

[0034]

Further , it is preferable to arrange a micro flow channel for a negative control.

Here, the term "a negative control" means a control for i ndi cat i ng a detection level ( a s mentioned above ) of the substrate itself in detecting DNA after treatment of D A ampl i fication reaction, so that the background intensity of the substrate is provided . That is, particularly when the DNA detection level is low, by comparing the micro flow channel of the target DNA with the negative control , it may be evaluated whether the detection level of each of micro flow channel indicates the amplified DNA or the background of the substrate.

Therefore, no primer is introduced to the micro flow channel of the negative control. According to such a configuration, even when the DNA extract is introduced to the micro flow channel of the negative control, and the temperature control a s ment ioned be 1 ow is conducted, no DNA ampl i fication take s place in the mi cr o flow channel of the negat ive control .

[0035]

Furthermore , it is preferred to provide a sealing means for sealing the open end of each of the micro flow channels, so that the DNA extract introduced thereinto may be prevented from the evaporation at the open end of the micro flow channel. Such sealing means may include any material to seal the open end, preferably a substance in a 1 iquid form in view of workability and the like. I n particular, a 1 i qu i d having high boiling point is preferable, since it does not vaporize under the heating condition in the DNA amplification reaction. Mo s t preferable is mineral oil, in view of handling.

[ 0036 ]

Here is illustrated a holder for holding the substrate a s an embodiment for employing the sealing means . FIG. 3 shows one embodiment of the holder . Fig . 4 shows a cross-section diagram of the holder .

[0037]

I n F I Gs 3 and 4, the substrate holder 50 is conf igured by an upper part 50a and a lower part 50b. The upper part 50a is provided with a guide 51a penetrating in a tapered shape through the upper part 50a for introducing the DNA extract into the substrate 40 , and a sealing material feed 52. The lower part 50b is provided with an opening 51b cylindrically penetrating through the lower part 50b. Further , between the guide 51a and the opening 51b, a holding part is provided for holding the substrate 40. Moreover , when the upper part 50a and the lower part 50b are brought into contact , an open space 53 for the sealing material appears adjacent to the holding part, so that the open space 53 for the sealing material has a contact with the outer circumference of the substrate 40 when holding the substrate 40. The sealing material feed 52 penetrates through the upper part 50a and is communicated with the open space 53.

[0038]

According to such a configuration, by separating the upper part 50a and the lower part 50b, holding the substrate 40 at the holding part of the substrate holder 50 in whi ch the feed 11 is faced toward the upper part 50a, and bringing into contact with the upper part 50a and the lower part 50b again, the substrate 40 is held in the substrate holder 50. Further , by dropping the DNA extract collected by the extract collecting means down to an opening of the guide 51a, the DNA extract is introduced into the mi cr o f 1 ow channel s 21a to 2 Ih through the feed 11 on the substrate 40. Moreover , mineral oil is introduced a s a sealing means through the sealing material feed 52 into the open space 53 for the sealing material , and f lows around the outer circumference of the substrate 40 , and into each of the micro f 1 ow channel s .

[0039]

I n FIG. 4 , it is illustrated that the shape of the opening 51b provided on the lower part 50b is in a cylindrical form . However , as shown in FIG. 5 , the lower part 50c may be employed which is provided with the opening 51c of a tapered shape. By conf igur ing the opening 51c in such form, it is hard to retain vapor and air on the opening, so that it is realized to control the temperature for the DNA ampl i fication reaction more accurately .

Further , in F I Gs . 4 and 5 , the guide 51a may be formed in a cylindrical form. However, by conf igur ing it in a tapered form a s shown in F I Gs . 4 and 5 , there is conveniently no shade in making an observation on the micro flow channel s after the DNA ampl i fication reaction is conducted in the substrate 40.

[0040]

I n FIG. 6 , the function of the sealing means is illustrated with the enlarged principal diagram of one mi cro f low channel and the open space 53 for the sealing mat er i al in vicinity thereof .

According to FIG. 6 , the mi cro f low channel 21a is filled with the DNA extract 31 in the substrate 40 being held by the substrate holder 50. On the other hand , the open space 53 for the sealing mat er i al has a contact with the end of the mi cro f low channel 21a. Mineral oil 33 being introduced as the sealing means f lows in the open space 53 for the sealing material , and then reaches to the outer circumference of the substrate 40. At this time , the mineral oil 33 flows in the micro f 1 ow channel 21a from the open end thereof . Accordingly, the open end of the micro f 1 ow channel 21a is sealed by the mineral oil 33 , resulting in the air layer 32 next to the mineral oil 33 in the mi cr o flow channel 21a, so that the DNA extract may be prevented from the evaporation by heating at time of the DNA ampl i fication reaction .

[0041]

A thermal cycler controls the heating condition for the DNA ampl i fication reaction in the micro flow channel s of the substrate . A thermal cycle in accordance with a temperature profile a s shown in FIG. 7 may be exempl i f i ed as the heating condition .

In FIG. 7 , the temperature profile is represented by the relationship of heating temperature to the e 1 ap s ed time . Spec ifically, the temperature profile includes a preheating period from room temperature ( RT ) to annealing temperature (T_L) ( t_i to t₀); an initial heating period from the temperature T_L to denaturing t emperatur e T_H (t₀ to 11 ) ; a denaturing period at the temperature T_H ( 11 to 1₂ ) ; a cooling period from the temperature T_H to the temperature T_L ( 1₂ to 1₃ ) ; an annealing period at the temperature T_L ( t₃ to t₄); a first heating period from the temperature T_L to DNA melting temperature (T_M) (t₄ to 15 ) ; an e 1 ongat i on reaction period at the temperature T_M ( 1₅ to 16 ) ; and a second heating period from the temperature T_M to the temperature T_H (t₆ to 1₇ ) . The thermal cycle of 11 to 1₇ is repeated by prescribed time s .

[0042] For example, in the thermal cycler, the temperature T_L is set to a temperature from 53 degrees C to (63 ± 0.5) degrees C; and the temperature T_M is set to a temperature from 70 to 72 degrees C; and the temperature of T_H is set to a temperature from 95 to 99 degrees C . Further , the period of the time is set a s follows : t_i to 1₀ , 5 seconds or less; 1₀ to t_lf 0.5 second or less 11 to 1₂ , 0.2 second or less; 1₂ to t₃ , 0.5 second or less; 13 to 1₄ , 0.2 second or less; t₄ to 1₅ , 1 second or less; 15 to 16 , 2.5 seconds or less; t₆ to 1₇ , 0.5 second or less. Moreover , the one total temperature cycle ( 11 to 1₇ ) is set to 5 seconds or less. Furthermore , the temperature cycle is repeated by at least 20 times, and preferably about 35 times.

[0043]

For realizing such temperature control for the DNA ampl i fication reaction, a steam thermal cycler ( heating and cooling apparatus ) is suitably employed, which is conf igur ed in a manner to control the temperature for the DNA amplification reaction by heating by steam and cooling by dry air the substrate .

[0044]

FIG. 8 shows a diagram illustrating the configuration of a steam heating and cooling apparatus as a thermal cycler in the embodiment of the invention . FIG. 9 shows a principal diagram illustrating the heating and cooling apparatus of FIG. 8. According to FIGs. 8 and 9, the heating and cooling apparatus 20 contains a motor 60 equipped with an axis 60a carrying a supporting board 60b for supporting the substrate 40 and being capable of rotating the supporting board 60b in the direction of the arrow R ; a steam nozzle 61 conf igured a s facing to the rotating trajectory of the substrate 40 by action of the motor 60 ; a steam generator 71 for generating and feeding steam to the s team nozzle 61 ; an air noz z le 62 configured as facing to the rotating tra ectory of the substrate 40 by action of the motor 60 ; a blower 72 for generating and feeding air having a lower temperature than the s team from the steam nozzle 61 to the air nozzle 62 ; an air nozzle 63 conf igured a s facing to the rotating trajectory of the substrate 40 by action of the motor 60 ; a blower 73 for generating and feeding air having a lower temperature than the air from the air nozzle 62 to the air nozzle 63 ; a control unit 70 for controlling the action of the motor 60 , the steam generator 71 , the blower 72 and the blower 73. Further , not shown in FIGs, the heating and cooling apparatus 20 is provided with a water supplier for supplying water for the steam to the steam generator 71.

[0045]

I n this embodiment , a steam f low of the saturated water vapor is empl oyed as the air f low at high temperature , and a dry air is employed a s the air f low at lower temperature than the steam and the airflow at further lower temperature . However, there is no limitation, as long as the substrate 40 may be heated or cooled at the prescribed temperature; that is, when pass i ng over the steam nozzle 61 , the substrate 0 is heated to the temperature T_H ; and when passing over the air nozzle 62 , the substrate 40 is heated ( or cooled ) to the temperature T_M ; and when pass i ng over the air nozzle 63 , the substrate 40 is cooled to the temperature T_L.

[0046]

The control unit 70 is suppl ied with electricity by an outer electrical source not shown in FIG. The steam generator 71 , and two of the blowers 72 and 73 is electrically connected with the control unit 70 , to thereby be suppl ied with the electricity and controlled .

[0047]

Here is i 1 lu s t rat ed the heating and cooling procedure of the substrate 40 by the heating and cooling apparatus 20 in accordance with the temperature profile a s shown in FIG. 7.

[0048]

(1) Attaching and supporting the substrate 40 on the end of rotating s ide of the support ing board 60b;

(2) Starting the steam generator 71 , and two of the blowers 72 and 72 by action of the control unit 70 , so that the substrate 40 is ready for sub ecting to heating or cooling at the preset temperature when pas sing over the corresponding nozzles; (3) Starting the motor 60 by action of the control unit 70, so that the substrate 40 supported by the supporting board 60b is moved over the air nozzle 63 to thereby be subjected to the preheating period (t_i to 1₀ ) , and thereafter moved over the steam nozzle 61 to thereby be sub ected to initial heating period ( 1₀ to ti ) , and maintained at that temperature for prescribed time ( 11 to 12 ) , so that the DNA denaturing occurs in the mi cr o flow channels of the substrate 40;

(4) Moving the substrate 40, passing over the air nozzle 62 to the upper place to the air nozzle 63 to thereby be sub j ected to the cooling period ( 1₂ to 1₃ ) , and maintained at that temperature for prescribed time ( 1₃ to t₄) , so that the annealing occurs in the mi cro flow channels of the substrate 40 ;

(5) Further moving the substrate 40 over the air nozzle 62 to thereby be sub ected to the first heating period (t₄ to

15 ) , and maintained at that temperature for prescribed time (t₅ to 16 ) , so that the DNA ampl i fication ( e 1 ongat i on ) reaction occurs in the mi cro flow channels of the substrate 40 ;

(6) Further moving the substrate 40 over the stream nozzle 61 to thereby be sub ected to the second heating period (t₆ to

17 ) , so that one cycle of the temperature control is over;

(7) Repeating the temperature control shown as the temperature profile of 11 to t₇, the above steps (3) to (6) for prescribed times, and then finally bringing the substrate 40 back to the position a s shown in F I Gs 8 and 9, heating and cooling procedure is terminated.

[0049]

I n the above step ( 3 ) , right after steam fed by the steam nozzle 61 reaches to the surface of the substrate 40, the s team become s concentrated to be minuscule droplet s of water, so that by the droplets of the high temperature the substrate 40 whi ch has the excellent thermal conductivity is rapidly heated from the temperature T_L to the temperature T_H , and maintained .

[ 0050 ]

I n the above step (4), when feeding the air from the air nozzle 63 to the substrate 40 , the concentrated and minuscule droplet s of water evaporate to thereby cool the substrate 40 from the temperature T_H to the temperature T_L, and maintained . I n the cooling process by the dry air , the minuscule droplet s of water can be rapidly evaporated, the substrate 40 can be cooled down quickly .

[0051]

I n the temperature control for the DNA ampl i fication reaction, the temperature of the substrate 40 may be measured by a thermometr ic means such a s a thermocouple to thereby change appropriately the heating or cooling condition at each of steps in accordance with the measured temperature , such as adjusting the steam temperature generated by the steam generator 71 , and adjusting the air temperature generated by the blowers 72 and 73 , and ad just i ng time for maintaining the substrate 40 above each of the nozzles.

[ 0052 ]

The conventional heating and cooling apparatus requires a continuous power supply, so that an external power supplier is generally used. On the other hand, according to the present invention, the heating or cooling time should be shorter, so that a battery or a portable second cell may be employed as a power source. Therefore, the heating and cooling apparatus could be portable, so that the apparatus could be used in other place than laboratory, in particular, out s ide . Further , the substrate is made from quar t z , so that the thermal conductivity is further improved, and then such an effect should increase .

[ 0053 ]

Further , the pathogen di agno s i s system may contain a spectrometer for spectroscopically detecting 1 abe 1 i ng agent which reacts with the produced DNA by the DNA ampl i fication reaction . Such spectrometer make s it possible to detect the DNA amplification reaction, even in the case of showing the spectroscopically 1 ow sensitivity because of shorting the DNA content in the body fluid .

[0054]

Since the DNA ampl i fication reaction is detected in the micro flow channel means that the primer which is allowed to be introduced therein in advance should i nvo 1ve the DNA ampl i fication reaction, so that it is sugge s ted that the DNA being reactive with the primer as a template DNA is included in the DNA extract from the body fluid. Therefore, it is recognized that a person providing the body fluid develops the infection involved by the pathogen ( s ) which should cor re spond to the primer ( s ) causing the DNA amplification reaction .

Accordingly, in the case that the DNA amplification reaction is detected, identifying the micro flow channel ( s ) and the primer ( s ) introduced therein makes it possible to identify the pathogen (s) which develops the infection in a person providing body fluid .

[0055]

From another aspect , the present invention provides a use of the pathogen diagno s i s system, that is, a diagno s i s method of pathogen . I n particular , the present invention provides a diagno s i s method of pathogen for identifying pathogen (s) i nvo 1ved in developing infection wherein there are a plural of pathogens which may be involved .

Specifically, as shown in FIG. 10 , the method comprises : a body fluid collecting step ( S 20 ) for collecting part of body fluid be i ng provided from a test body and containing DNA; a DNA extraction step ( S 30 ) for extracting DNA from the collected body fluid to obtain DNA extract ; a DNA ampl i fication reaction step (S40) for conducting DNA ampl i fication reaction in micro flow channel s being configured in a substrate containing at least the s ame number of micro flow channels a s the number of pathogens or of groups of pathogens to be detected, in the micro flow channels the DNA extract obtained in the step ( S 30 ) , and the PGR reagent including thermostable DNA polymerase , buf f er , deoxynucleoside triphosphates, and labeling agent being spectroscopically detectable by reacting with the amplified DNA being loaded, and primers being introduced prior to the DNA ampl i fication reaction in each of the micro flow channel s in a manner that one kind of primer s is ne er introduced in two or more micro flow channels, corresponding to each of a plural pathogens and being for amplifying each of target DNAs having sequence distinguishing each of the pathogens ; and a detecting step ( S 50 ) for detecting labeling agent which reacts with the produced DNA by the DNA amplification reaction, wherein the pathogen corresponding to a primer being loaded in the micro flow channel in which the labeling agent is detected in the detecting step ( S 50 ) is identified a s a pathogen involved in developing infection ( S 60 ) .

[ 0056 ]

I n FIG. 10 , in the step ( S 10 ) , a substrate is prepared in a manner that primers are introduced in mi cro flow channel s . I n particular, a s mentioned above , the primer reagent s are introduced from prescribed positions of the substrate corresponding to each of the micro f 1 ow channel s , and then the substrate is sub ected to drying . At this time, it must be noted that one primer should not be introduced in two of more micro flow channels; that is, each of the micro flow channels contains the different primer(s) from one another.

[0057]

I n the step ( S 20 ) , a body fluid (for example , muco sal fluid ) of a patient a s a test body is sampled by a body fluid collecting means .

[ 0058 ]

I n the step ( S 30 ) , the body fluid sampled in the step ( S 20 ) is introduced and mixed in a DNA extraction container with a DNA extraction reagent , to thereby conduct the DNA extraction reaction and obtain a DNA extract . To the DNA extraction container may be introduced together a PGR reagent together prior to the introduction of the body fluid a s ment ioned above . Further , in the DNA extraction reaction, the DNA extraction container into which the DNA extraction reagent and the body fluid are introduced may be heated or cooled ( or regulated ) , so that the reaction may be initiated, promoted or terminated .

[ 0059 ]

I n the step ( S 40 ) , the DNA extract obtained in the step ( S 30 ) is collected by use of an extract collecting means , and then introduced from a feed for the extract into the micro flow channel s conf igured on the substrate be i ng prepared in the step ( S 10 ) . Thereafter , the heating and cooling condition for the substrate is controlled in a manner that the temperature profile a s shown in FIG. 7 is repeated for prescribed time s , so that the micro flow channels are subjected to the condition for amplifying DNA.

Further , at the time , one of the micro flow channels may be used as a positive control. Furthermore, one of the rest of the mi cro flow channels may be used a s a negative control .

In controlling the heating and cooling conditions, a steam thermal cycler (heating and cooling apparatus) may be used, so that heating by steam and cooling by dry air for the substrate are conducted.

[0060]

I n the step ( S 50 ) , the detection is conducted for r ecogni zing if the ampl i fication reaction is occurred by the heating and cooling operation on the substrate being conducted in the step ( S 40 ) ; that is, the labeling agent which is reacted with the ampl i f i ed DNA is detected . Detecting the labeling agent ( s ) indicates that the DNA ampl i fication reaction takes place in the detected micro flow channel ( s ) . Further , in the detection, a spectrometer may optionally be used .

[0061]

In the step ( S 60 ) , the micro flow channel (s) in which the

1 abe 1 i ng agent is detected in the step ( S 50 ) is ident i f ied , so that the di agno s i s is conducted by identifying the pathogen ( s ) , corresponding to the primer ( s ) which is introduced in the detected micro f low channel , a s pathogen ( s ) i nvolved in developing infection in the test body who provides the body fluid. Then, the diagnosis is finished. If no labeling agent is detected from any micro flow channel, the diagno s i s is conducted to r e cogni z e that no pathogen, corresponding to any of primer being introduced in the micro flow channel, involves in developing infection in the test body .

[ 0062 ]

From further aspect, the present invention provides a DNA amplifying kit which is suitably used for the pathogen diagno s i s system, in particular , a DNA amplifying kit for amp 1 i fy i ng a spec i f i c target DNA ( s ) in parallel which exists in a s amp 1 e containing DNA.

Spec ifically, the kit comprises : a s ampl e collecting means for collecting part of a s ampl e containing DNA, such as a body fluid from a patient with infection ; a DNA extraction reagent for extracting DNA from the collected sample to obtain DNA extract; a PGR reagent for amplification reaction of DNA on the DNA extract, comprising thermostable DNA polymerase, buf f er , deoxynucleoside triphosphates, and labeling agent being spectroscopically detectable by reacting with the ampl i f i ed DNA; a plural types of primer reagents each comprising one or more kinds of primers for amplification of target DNAs, each primer corresponding to each of the target DNAs ; a DNA extraction container for providing a field for DNA extraction reaction by use of the DNA extraction reagent ; an extract collecting means for collecting part of DNA extract obtained in the DNA extraction container; and a substrate containing at least the same number of micro flow channels as the number of the target DNAs or of groups thereof , in the mi cro flow channels the DNA extract collected by the extract collecting means, the PGR reagent and the primer reagent being loaded, and a field for DNA amplification reaction being pro ided , wherein primer ( s ) , that is, a primer or a group of primers is allowed to be introduced in each of the micro flow channel s , and wherein one primer is not introduced in two or more micro f low channel s . Further , the kit may contain an instruction for guiding an operator in such a way of operating the kit in a protocol in the order of the steps ( S 10 ) to ( S 40 ) , and steps ( S 50 ) and ( S 60 ) , as shown in FIG. 10 , if needed .

Moreover, when the kit is used in a di agno s i s for infection, in whi ch the pathogen ( s ) to be sub ected to the detection, and DNA ( s ) to be amplified is predetermined, the kit may be provided in such a manner that the primer reagent ( s ) is introduced in advance in mi cro f low channe 1 ( s ) of the substrate .

Accordingly, the operator only operates the kit in starting with the step ( S 20 ) of FIG. 10 , so that the ampl i fication of DNA and diagnosis for identifying the pathogen ( s ) of infection may be conducted . I n such a case , the kit may contain an instruction for guiding an operator in such a way of operating the kit .

EXAMPLES

[ 0063 ]

The present invention is explained in greater detail using the examples below. The present invention is not limited to these examples.

[0064]

( Example 1 )

The following bacteria a s known for typi cal pathogenic bacteria of pneumonia are focused. DNA fragment s a s shown in the following t able corresponding to each of the bacteria (both of "FORWARD" direction amplification and "REVERSE" direction ampl i fication of the DNAs specific to each of the bacteria ) were employed a s suitable primers for DNA ampl i fication by the PGR method of those bacteria .

[0065]

Table 1

MSSA: Methicillin-Sensitive Staphylococcus Aureus

MRSA: Methicillin-Resistant Staphylococcus Aureus

0066 ]

A substrate of borosilicate glass configured a s shown in FIG. 1 (CAs-CHIP; manufactured by METABO SCREE CO., LTD.) was sterilized. Thereafter, to each of seven micro flow channels out of eight were separately introduced the primers (both of FORWARD and REVERSE) corresponding to each of the bacteria as shown in Table 1 , respectively . To the rest of one micro f 1 ow channel was introduced a primer pair whose target gene is human bet a-gl obi n (bglo-F&R) for exhibiting the positive control . Each reagent was set in the eight of micro flow channels, and the substrate was subjected to drying, to thereby obtain a primer-containing substrate in which the primers were introduced into the micro f 1 ow channel s .

The dimension of the substrate was 10 mm in diameter and 0.3 mm in thickness; and the dimension of each of the mi cro flow channel s was 5 mm in length, 0.1 mm in width and 0.1 mm in depth .

[0067]

( Example 2 )

From the test body who developed pneumonia, one platinum loop of purulent sputum was sampled. The sampled purulent sputum was mixed in a tube together with CellEase ( tm ) I I

( available from Biocosm Inc . ) used as a DNA extraction reagent . I n the tube , prior to introducing the purulent sputum, a PGR reagent containing thermostable DNA polymerase and buf fer for reaction ( TaKaRa Ex Taq ( tm) , available from TAKARA BIO INC . ) and a spectroscopically detectable 1 abe 1 i ng agent (SYBR ( tm ) Green I, available from TAKARA BIO INC.) were already mixed.

[0068]

( Example 3 )

The DNA extract thus obtained in Example 2 was dropped onto a feed for the extract of the primer-containing substrate prepared in Example 1, to thereby be introduced into each of the mi cro flow channels . Subsequently, the substrate was subjected to the heating and cooling treatment by a steam heating and cooling apparatus as shown in FIGs. 8 and 9 under the condition for repeating 35 time s of a thermal cycle wherein in accordance with the temperature profile a s shown in FIG. 7 the annealing temperature (T_L) was set to 60 degrees C ; the ampl i fication temperature ( T_M ) to 72 degrees C ; and the denaturing temperature ( T_H ) to 95 degr ee s C ; each period of time was set to the following table .

[0069]

Table 2

[0070]

( Example 4 )

The substrate which was subjected to the condition for the DNA amplification reaction in Exampl e 3 was irradiated with an exciting light at the center of the wavelength of 450 nm ( cut from 500 nm or longer ) and irradiation area of 10 mm s quare or larger , and then with a detecting light at the center of the wavelength of 520 nm ( cut from 500 nm or shorter ) , and then took an image of the surface of the substrate , to thereby detect if the DNA ampl i fication reaction took place .

[0071]

( Example 5 )

Since the DNA ampl i fication reaction was detected in Exampl e 4 at the micro f 1 ow channel in which the primer corresponding to Staphylococcus aureus (MRSA) had been introduced, Staphylococcus aureus ( MRSA ) was identified as a pathogen involved in developing pneumonia. EXPLANATIONS OF LETTERS OR NUMERALS

[0072]

11, lib : Feed for DNA Extract

11a: Primer Feed

20 : Heating and Cooling Apparatus

21a to 21h: Micro Flow Channel

22a to 22s: Micro Flow Channel

23a to 23s: Micro Flow Channel

31 : DNA Extract

32 : Ai r Layer

33 : Mineral Oil

40, 41: Substrate

50 : Substrate Holder

51a: Guide

51b, 51c: Opening

52 : Sealing Material Feed

53 : Open Space for Sealing Material

60: Motor

60a: Axi s

60b: Supporting Board

61 : Steam Nozzle 62, 63: Air Nozzle

70 : Control Unit

71: Steam Generator

72, 73: Blower

δ

0

5 SEQUENCE LISTING

<110> Daiken Medical Co., LTD

<120> DNA AMPLIFYING KIT <130> TEK/FP6882161

<160> 14

< 170 > Patentln version 3.5

<210> 1

<211> 21

<212> DNA

< 213 > Artificial Sequence <220>

<223> Primer for PCR

<400> 1

caaccgtaca gaatgaagcg g

21

<210> 2

<211> 22

<212> DNA

<213> Artificial Sequence <220>

<223> Primer for PCR

<400> 2

ttattcgtgc aatactcgtg eg 22 <210> 3

<211> 21

<212> DNA

<213 > Artificial Sequence <220>

<223> Primer for PCR

<400> 3

ttgacatcct aagaagagct c 21

<210> 4

<211> 21

<212> DNA

<213> Artificial Sequence <220>

<223> Primer for PCR

<400> 4

tctcctttga gttcccgacc g 21

<210> 5

<211> 21

<212> DNA

<213 > Artificial Sequence <220>

< 223 > Primer for PCR <400> 5 gcgattgatg gtgatacggt t 21

<210> 6

<211> 25

<212> DNA

< 213 > Artificial Sequence <220>

<223> Primer for PCR

<400> 6

agccaagcct tgacgaacta aagcc 25

<210> 7

<211> 21

<212> DNA

< 213 > Artificial Sequence <220>

<223> Primer for PCR

<400> 7

tatccaccct caaacaggtg a 21

<210> 8

<211> 21

<212> DNA

<213> Artificial Sequence <220> <223> Primer for PGR

<400> 8

ctggaacttg ttgagcagag g 21

<210> 9

<211> 21

<212> DNA

< 213 > Artificial Sequence <220>

<223> Primer for PGR

<400> 9

atacagtggt cagtgatcgc c 21

<210> 10

<211> 21

<212> DNA

< 213 > Artificial Sequence <220>

<223> Primer for PGR <400> 10

agaagtacca ttaccaccgc c 21

<210> 11

<211> 22

<212> DNA <213> Artificial Sequence <220>

<223> Primer for PGR

<400> 11

cacttcatcg gctacatcta eg 22

<210> 12

<211> 22

<212> DNA

< 213 > Artificial Sequence <220>

< 223 > Primer for PGR

<400> 12

agatattege tctggtaggt gg 22

<210> 13

<211> 20

<212> DNA

<213> Artificial Sequence <220>

<223> Primer for PGR

<400> 13

caaccgatgc cacatcatta 20

<210> 14 <211> 20

<212> DNA

< 213 > Artificial Sequence <220>

<223> Primer for PGR

<400> 14

atagcgtctt gcatgccttt 20

Claims

What is claimed:

1. A DNA amplifying kit for amplifying a specific target DNA ( s ) in parallel which exists in a sample containing DNA, comprising :

A sample collecting means for collecting part of a sample containing DNA ;

A PCR reagent for ampl i fication reaction of DNA on the DNA extract , compr i s i ng thermostable DNA polymerase , buffer , deoxynucleoside triphosphates, and labeling agent being spectroscopically detectable by reacting with the ampl i f i ed DNA;

A plural types of primer reagents each compr i sing one or more kinds of primers for amplification of target DNAs , each primer corresponding to each of the target DNAs ;

A DNA extraction container for pro iding a field for DNA extraction reaction by use of the DNA extraction reagent ;

An extract collecting means for collecting part of DNA extract obtained in the DNA extraction container ; and

A substrate containing at least the same number of mi cro f low channel s as the number of the target DNAs or of groups thereof, in the mi cro f low channels the DNA extract collected by the extract collecting means , the PCR reagent and the primer reagent being loaded, and a field for DNA ampl i fication reaction being provided,

Wherein primer ( s ) is allowed to be introduced in each of said micro flow channels; and

Wherein one primer is not introduced in two or more micro 5 f low channels .

2. The kit a s claimed in Claim 1 , wherein said sample collecting means is made up of an element formed in a manner to hold said sample and remove the moisture of the sample , and an element for collecting the dehydrated sample .

3. The kit a s claimed in any of Claim 1 or 2 , wherein said substrate further comprises a micro f low channel for a positive control .

δ

4. The kit as c 1 aimed in Claim 3 , wherein a template

DNA having the different sequence from any of the target DNAs and a primer having the sequence complementary to a part of the sequence of said template DNA are introduced in advance0 to said micro f low channel of the positive control .

5. The kit as c 1 aimed in Claim 3 or , wherein said substrate further compri ses a micro flow channel for a negat i ve control .

5

6. The kit as claimed in any of Claims 1 to 5, wherein the substrate is provided with a feed for introducing the DNA extract ; and wherein each of micro f 1 ow channels is conf igured in a manner that one end of each channel s connects with said feed of said substrate and the other end of each channel s is open , respectively .

7. The kit a s c 1 aimed in Claim 6 , further compr i s i ng a sealing means for sealing the open end of each of the micro flow channel s .

8. The kit a s claimed in Claim 7 , wherein said sealing means is mineral oil.

9. The kit a s claimed in any of Claims 1 to 8 , wherein said primer reagent ( s ) is pr e 1 imi nary introduced in said mi cro f low channe 1 ( s ) of said substrate .

10. The kit a s claimed in any of Claims 1 to 9 , further compr i sing an instruction for guiding an operator in operating said kit.