WO2024028886A1

WO2024028886A1 - A miniature quantitative polymerase chain reaction apparatus

Info

Publication number: WO2024028886A1
Application number: PCT/IN2023/050262
Authority: WO
Inventors: Jatin Singh Banga; Nilesh Hukmichand Singhvi; Trushal Balkrishna Alshi; Rajat CHATURVEDI; Amit Rajendra Prasad Pal
Original assignee: Biosense Technologies Pvt. Ltd.
Priority date: 2022-09-09
Filing date: 2023-03-18
Publication date: 2024-02-08

Abstract

The present invention discloses a miniature quantitative polymerase chain reaction (qPCR) apparatus. A qPCR works on the principle of amplification of nucleic acids from a small sample. The efficiency and uniformity of the nucleic acid amplification is therefore the key to an accurate Polymerase Chain Reaction. Thus, the largest challenges of a PCR apparatus are achieving accurate readings typically affected by the efficiency and uniformity of amplification especially when considering an apparatus designed for a smaller size and to be operated by non-skilled persons. The improved miniature quantitative polymerase chain reaction apparatus of the present disclosure relates to a compact apparatus while ensuring accuracy in processing sample in real time. The miniature quantitative polymerase chain reaction apparatus of the present invention minimizes the interferences of light noise and errors in results caused by shifting of the optical filter(s) while offering a compact and easy to operate apparatus. The improved apparatus of the present invention therefore overcomes the shortcomings of the existing qPCR apparatus.

Description

A MINIATURE QUANTITATIVE POLYMERASE CHAIN REACTION APPARATUS I) OF INVENTION:

The present invention in general, relates to a quantitative polymerase chain reaction. Particularly, the present invention relates to a miniature quantitative polymerase chain reaction apparatus offering high accuracy of results and overcoming the need for skilled persons and laboratory environment to operate said apparatus.

BACKGROUND OF THE INVENTION

Polymerase chain reaction (PCR) is a method widely used to synthesize billions of copies of asample genetic material (DNA). This method is used by skilled laboratory persons for a variety of tasks requiring DNA amplification, examples of such tasks include detecting hereditary diseases, cloning of genes, paternity testing, DNA computing, etc.

In a typical PCR process, the sample DNA is separated into its two strands, a primer is used to synthesize copies of each strand doubling the DNA. This process is repeated until the PCR process exponentially amplifies DNA. Amplification of genetic material is crucial to detecting and analyzing the DNA molecules.

For the PCR process to work ideally periodic temperature changes or temperature cycling is used to generate billions of copies of a target nucleic acid sequence from limited starting amounts of nucleic acids. Generally during PCR, target DNA is amplified by denaturing the DNA (occurs at high temperatures ~95C), annealing (occurs at low temperatures ~60C) short primers to resulting single strands at specific sites (e.g., sequences flanking the target site) andextending the primers using a thermostable polymerase to generate new copies of double- stranded DNA complementary to the target, the formation of new copies offer fluorescence asan indicator to measure the amount of newly formed DNA. The detection of this fluorescence requires an optical body to measure the intensity and thereby quantify the amount of newly formed DNA. Typically, the PCR reaction mixture is repeatedly cycled (e.g., 20-50 times) from high temperatures (e.g., >90° C.) to denature the DNA to lower temperatures (e.g., between about 37° C. to 70° C.) for primer annealing and extension. Primer annealing and extension can be performed at the same or different temperatures. However, the PCR process is sensitive to sample size and temperature, meaning different sample sizes requires slightly different temperature ranges and unless such temperatures are accurately achieved a large difference inthe final amplification is observed.

In most automated PCR instruments, the reaction mixture is placed within a disposable plastic tube which is closed with a cap and placed within a metal heat-conducting sample block. The sample block is in communication with a processor which controls the cyclical heating of the block. As the metal block changes temperature, the reaction mixture is exposed to similar changes in temperature. The use of these types of heating devices can result in delays in transferring heat from the sample block to the reaction mixture which may not be the same for all samples. Thus, both the efficiency and uniformity of amplification of nucleic acids within the samples can suffer as a consequence.

Evaporation from the PCR mixture during thermal cycling also can decrease the uniformity of amplification. Since the reaction mixture generally occupies only a fraction of a sample tube, this leaves a volume of air (known as “head space”) above the reaction mixture into which thereagents of the reaction can evaporate and subsequently condense.

Based on the conventional PCR, is a real-time quantitative PCR (qPCR). In general qPCR instruments work in “real-time” detecting newly made DNA by quantifying the fluorescence in a fluorescent labelled probe using 2 modules: a) A temperature cycling and controlling system and b) An optical body for fluorescence detection/monitoring system. An optical body as disclosed above is employed to measure the intensity of the fluorescence emission from eachof the sample tubes, to quantify the amount of newly formed DNA. To measure fluorescence, an excitation light is directed at the samples in the sample wells, on excitation, the light emittedfrom the fluorophores in the samples (newly formed DNA) is detected. Thus, for an accurate reading it is crucial that the light transferred from the light source to the wells be carried effectively and efficiently with minimal wastage. Often the close proximity of wells results inthe mixing of light in transit especially when using more than one light source for multiple andsimultaneous sample well detection.

Optical systems for directing light for sample detection in PCR are disclosed by US 6,942,837 and US 7,410,793. However, the need for desired accuracy to be met with optimal carrying oflight still persists.

Additionally, environmental deterrents such as stray light and pollutants also play a role in altering the accuracy of the fluorescence which affects the results of the PCR apparatus. Furthershortcomings of currently available qPCR apparatus include the lack of a miniature apparatuswhich can perform a real-time qPCR function with desired accuracy.

There have been numerous attempts towards achieving an apparatus that can offer accurate results, however, these apparatuses are very large, complex, require skilled persons & laboratory environment to operate and are mostly inoperable in remote locations. Therefore, there is a need for a qPCR apparatus that can overcome at least one of the above mentioned problems. SUMMARY OF THE INVENTION

Before the present apparatus, is described, it is to be understood that this application is not limited to the particular disclosure, and details described, as there can be multiple possible embodiments which are not expressly illustrated in the present disclosure. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only and is not intended to limit the scope of the present application. This summary is provided to introduce concepts related to miniature quantitative polymerase chain reaction apparatus described further below in the detailed description. This summary is not intended to limit essential features of the subject matter nor is it intended for use in limiting the scope of the subject matter.

An aspect of the present invention relates to a miniaturized polymerase chain reaction apparatus which comprises of: a) A thermal cycler unit to cyclically and periodically regulate temperature within theapparatus; b) an optical body unit having at least one optical module to detect fluorescence within a sample housed within the thermal cycler; and c) a processing unit to control and communicate with units within the apparatus;

Another aspect of the present invention relates to a miniaturized polymerase chain reaction apparatus characterized in that the optical module of the optical body, has a notch to secure atleast one optical filter at a 45° angle for optimum detection of fluorescence in sample.

Yet another aspect of the present invention relates to a miniaturized polymerase chain reaction apparatus having a thermal cycler which comprises of: at least a sample block, a Peltier, a heatsink and thermal pad(s) sandwiched between the Peltier and sample block, wherein the sample block contains up to 16 sample wells to contain sample.

Yet another aspect of the present invention relates to a miniaturized polymerase chain reaction apparatus having a sample block covered by an Aluminum covering and a Polyamide or siliconheater to reduce condensation.

Yet another aspect of the present invention relates to a miniaturized polymerase chain reaction apparatus having a thermal cycler made up of copper having a set of equally distanced fins.

Yet another aspect of the present invention relates to an optical body which comprises an excitation unit and a detection unit, at least one gasket ring at the excitation and the detection unit respectively, a mobile shaft and a plurality of optical modules.

Yet another aspect of the present invention relates to a miniaturized polymerase chain reaction apparatus having a PCB operated software which controls the temperature and detection in the PCR apparatus.

Yet another aspect of the present invention relates to a method of operating a miniaturized polymerase chain reaction apparatus

BREIF DESCRIPTION OF DRAWING

The foregoing detailed description of embodiments is better understood when read in conjunction with the appended drawings. For the purpose of illustrating the disclosure, example constructions of the disclosure are shown in the present document, however, the disclosure is not limited to the specific apparatus and method disclosed in the documents and the drawings. A detailed description is given with reference to the accompanying figures.

Figure 1 illustrates parts of the qPCR apparatus in accordance with an exemplary embodiment of the present invention.

Figure 2 illustrates a perspective view of the thermal cycler in accordance with an exemplary embodiment of the present invention.

Figure 3 illustrates a sectional view of the sample block in accordance with an exemplary embodiment of the present invention.

Figures 4 illustrate parts of the heat lid assembly in accordance with an exemplary embodiment of the present invention.

Figure 5a illustrates a detailed sectional view of the optical body in accordance with an exemplary embodiment of the present invention.

Figures 5b illustrates an external view of the optical body in accordance with an exemplary embodiment of the present invention.

Figure 6 shows the graphs resulted in experiments conducted to establish the accuracy and efficiency of the present invention in accordance with an exemplary embodiment of the present invention.

DETAIT, ED DESCRIPTION OF THE INVENTION

Some embodiments of this disclosure, illustrating all its features, will now be discussed in detail. The words “comprising”, “having”, “containing” and “including”, and other forms thereof, are intended to be equivalent in meaning and be open ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items. It must also be moted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Although any systems and methods similar or equivalent to those described herein can be used in the practice, the exemplary, systems and methods are now described. The disclosed embodiments are merely exemplary of the disclosure, which may be embodied in various forms. Industry specific terms such as quantitative polymerase chain reaction/ qPCR/ real-time PCR/ quantitative PCR shall be read in a non-limiting and an all-encompassing manner in the document.

Various modifications of the embodiment will be readily apparent to those skilled in the art and the generic principles herein may be applied to other embodiments.

However, one of ordinary skill in the art will readily recognize that the present disclosure is not intended to be limited to the embodiments illustrated but is to be accorded the widest scope consistent with the principles and features described herein.

The term “qPCR” is used interchangeably with “quantitative polymerase chain reaction”, both having the same meaning.

Various embodiments of the present invention relate to an improved miniature quantitative polymerase chain reaction apparatus (100). The qPCR apparatus as disclosed comprises components like Optical body (300), thermal cycler (200), Controller (427), inter alia.

In an embodiment of the present invention the miniature quantitative polymerase chain reaction apparatus (100) is disclosed, the apparatus comprising: a.) a thermal cycler unit (200) to cyclically and periodically regulate temperature within the apparatus (100); b.) an optical body unit (300) having at least one optical module (313) to detect fluorescence within a sample housed within the thermal cycler (200); and c.) at least a processing unit (400) to control and communicate with units within the apparatus;

Wherein, the apparatus (100) is characterized such that the optical module (313) of the optical body (300), has a notch (314) to secure at least one optical filter (315) at a 45 > angle for optimum detection of fluorescence.

In another embodiment of the present invention the thermal cycler unit (200) comprises: at least a sample block (205), a Peltier (206), a heat sink (207) and thermal pad(s) (212) sandwiched between the Peltier (206) and sample block (205), wherein the sample block contains up to 16 sample wells (201) to contain sample. The thermal cycler (200) is designed to maintain the heat during heating cycle, wherein the temperature prevents condensation of the sample inside the sample tube (202).

In another embodiment, the thermal cycler unit (200) houses a sample block having a heat lid assembly (500), sample tubes (202) containing sample placed within uniformly spaced sample wells (201) at a distance of 3mm - 6mm from each other and preferably at a distance of 3 mm - 4 mm measured between any two adjacent sample wells.

In yet another embodiment the thermal cycler unit (200) houses a heat lid assembly (500) measuring 106mm X 48mm X 32mm having at least one metal covering wall (211), a lid (203) and a heater (210) present over at least the top side of the sample wells to prevent condensation. Wherein, the thermal cycler unit houses a heater and a heat lid assembly having a metal lid made of Aluminium (203); and wherein the heater (210) is a polyamide heater. The metal covering wall made of Aluminum maybe on more than one side of the sample wells, effectively forming a casing around the sample wells as seen in Fig. 4. Another embodiment of the present invention discloses the thermal cycler (200), wherein the thermal cycler unit is made of copper and has equally distanced fins.

A further embodiment of the present invention discloses a miniature qPCR apparatus which comprises an optical body, wherein the optical body unit (300) comprises at least one optical module (313) having at least an excitation unit (320) to excite sample; at least a detection unit (322) to detect sample fluorescence post sample excitation; and a mobile shaft (326) to transport the optical module (313) from one well to another. The optical module houses at least one optical filter (315) which may be selected from a group comprising of a beam splitter optical detection system, a hybrid optical detection system, bifurcated optical detection system, and is preferably a beam splitter optical detection system.

The optical body of the present invention has at least one gasket ring (323) present at the detection unit (322) and at least on gasket ring (321) present at the excitation unit (320) to prevent any seepage of light.

In yet another embodiment, the present invention discloses the optical body (300) comprising: at least a detection unit, an excitation unit, a mobile shaft (326) and at least one optical module (313) having an optical filter (315) fixed within a notch (314) to maintain a 45° position, and wherein the optical filter is sandwiched between a top gasket (318) touching the top block (316)and a bottom gasket (319) sitting within the notch (314) on the bottom block (317). Wherein the optical filter(s) (315) is maintained at a constant distance from the object lens at any given point. The notch of the optical module ensures that the optical filter is locked within a specificposition; the gaskets further tighten the placement of the optical filter as well as prevent any cross-talking / seepage of light. Further, the gaskets reduce the wear and tear of the optical filter as the gaskets buffer the physical contact between the optical filter (315) and the top and bottom parts of the optical module. The top block (316) comprises of emission lens and filter and bottom block (317) comprises excitation and objective Lens and filters of specific led.

Another embodiment of the present invention discloses a miniature quantitative polymerase chain reaction, wherein the optical body (300) has two or more optical modules (313).

Yet another embodiment the present invention discloses a processing unit (400), which has a PCB operated by a software to at least control temperature of the quantitative polymerase chain reaction apparatus (100) and processing outputs of the detectors for analysis.

A thermal cycler (200) having a sample block (205) housing up to 16 sample wells (201) to hold sample tubes (202) containing sample to be detected; a lid (203) to cover the wells (201); an aluminium cover with at least one wall (211); a polyamide heater (210); a peltier (206); a heat sink (207); thermal pads (212) sandwiched between the peltier (206) and heat sink (207) with fins (208); and

An optical body (300) having at least one optical module (313) comprising a bottom block (317) having an object lens (325) excitation unit (320) securely lined by excitation gasket ring (321) to prevent any light noise/seepage; top block (316) having detection unit (322); said detection unit securely lined by detection gasket ring (323) to prevent any light noise; atleast one notch (314) to hold at least one optical filter (315) at a 45° angle wherein the notchis located between the top block (316) and the bottom block (317) sandwiched between the top gasket (318) and bottom gasket (319) to fixedly hold the optical filter (315) in position and to act as a cushion preventing any wear and tear due to direct contact by the top & bottom blocks. As light is emitted from the emission unit (322) it travels through the object lens (325) into the sample wells and onto the sample (housed within the thermal cycler) which remmits thefluorescence through the optical filter (315) to the detection unit (322).

EXAMPLES

Examnle 1:

Part 1 : Sample Preparation

1. A specimen using extraction kit was prepared using Amplichain- universal extraction kit as per detailed protocol on package inserts.

2. Resulting extracted specimen was used as starting sample for amplichain qPCR.

3. Total reaction mixture measured ~20uL.

4. Monotest type CMV kit was used in the first test, similarly DEN/CHIK kit was used in following tests to analyze specimen.

5. PCR tube having lyophilized master mix reagent was reconstituted using lOuL of reconstitution buffe refer as sample tube.

6. lOuL of extracted specimen was added to sample tube.

Part 2: Real-time PCR

1. Sample tubes were prepared and placed on the sample block to carry the qPCR reaction.

The sample tubes were sealed to prevent spillage or cross-contamination.

2. A PCR cycle was performed. The setting parameters changed as per the tests (CMV/ DEN/CHIK etc.). For example, for CMV detection the initial denaturation was carried out at 95-degree temperature for 3 min. Further, annealing was carried out at 95-degree temperature for 15 sec. Once done with annealing, the sample was subjected to extension at 55-degree temperature for 25 sec/min. The same process was carried for 45 cycles.

3. Likewise for DEN/CHIK detection different protocol needed to be followed. Den/Chik samples having RNA as a genetic material needed to be converted to cDNA by following extra Reverse Transcriptase step before going to actual PCR cycle. RT was carried out at 45-degree temperature for 15 min. Then followed by initial denaturation at 95-degree temperature for 3 min. Further, annealing was carried out at 95-degree temperature for 15 sec. Once done with annealing, the sample was subjected to extension at 55-degree temperature for 25 sec/min. The same process was carried for 45 cycles.

Part 3: Detection and Analysis

4. The LED or other light source for FAM and HEX channels were activated to stimulate fluorescence.

5. As the amplification was being carried out in sample tube fluorescence generated.

Fluorescence intensity was directly proportional to the copy number of nucleic acid within the reaction.

6. Simultaneously generated fluorescence was detected by the optical module which was mounted with LED (Fam/hex/Cy5) with particular emission of light.

7. The amplification curve was generated in positive sample tube with sigmodal curve shape, while, since there was not any amplification in negative sample tube, fluorescence was a straight line as shown in figure 6. ADVANTAGES OF THE INVENTION:

A few advantages of the miniature quantitative polymerase chain reaction of the present invention are disclosed here below. It is stated here that the below mentioned list is non- limitingand is provided to give a broad understanding of the novel properties of the present invention without limiting its scope or understanding.

1. The present invention offers a miniaturized quantitative polymerase chain reaction apparatus capable of delivering accurate results.

2. The miniature qPCR of the present invention overcomes the need of a skilled person tooperate the apparatus or the need of laboratory environments for the apparatus to function

3. The miniature qPCR of the present invention overcomes the shortcoming of unsecured positioning of the optical filter, such unsecured positioning may include an altered angle at which the optical filter is positioned at the time of operating the apparatus, it may also include an altered distance of the optical filter from the object lens, both of which may severely affect the results, this chance of error has been overcome by the present invention by at least including a notch and additionally by sandwiching the optical fibers using rubber gaskets.

4. The miniature qPCR of the present invention allows for operation of the apparatus in mobile location such as portable labs or testing centers created in vehicles.

5. The qPCR apparatus of the present invention reduces condensation of the sample inside the sample tube and thus reduces inaccuracy in detection of fluorescence.

6. The optical body houses rubber rings and rubber gaskets to avoid any seepage of light from affecting the readings of the emission detection. 7. The present invention offers a miniaturized quantitative polymerase chain reaction apparatus which is hardy, miniature, robust and accurate especially when compared to qPCRs of the market.

EXPERIMENTAL DATA:

ACCURACY AND EFFICIENCY:

The challenges faced in developing the present invention focused on Accuracy and Efficiency as these are most important requirements for any qPCR apparatus, the challenge was to ensure at least similar accuracy and efficiency in the miniaturized apparatus as compared to the regularly available (large) qPCR apparatus, one of the major interferences in achieving said accuracy was the “background noise” which may be caused due to seepage / cross-talk of light during the PCR run. Another major interference was the unstable position of the optical fiber which affected the accuracy every time it shifted, the shifting in the present invention was a larger challenge to achieve as the present invention is meant to be used in non-lab environment which are likely to have mobile conditions.

On developing the apparatus as per embodiments of the present invention, comparative tests were performed to measure accuracy and efficiency of the miniature qPCR of the present invention and results were compared with another qPCR device known as Reference qPCR Apparatus (known to have accurate results, but is not miniature in size) to comparatively test the accuracy levels of the present invention. The test involved a PCR run carried out on both machines using the same sample.

It was observed that the miniaturized qPCR of the present invention was found to meet the expected Ct value ranges for the test samples. Though, being a compact, mobile and miniaturized apparatus, the results were surprisingly more accurate than Reference qPCR Apparatus and thus the machine was proved to be more efficient. Below table shows the efficiency of the present invention. Moreover, Figure 6 shows an accurate result from the miniaturized qPCR apparatus, minimizing the background noise as compared to the results showing higher background noise when using Reference qPCR Apparatus.

Claims

CLAIMS A miniature quantitative polymerase chain reaction apparatus, comprising: a.) a thermal cycler unit (200) to cyclically and periodically regulate temperature within the apparatus; b.) an optical body unit (300) having at least one optical module (313) to detect fluorescence within a sample housed within the thermal cycler (200); and c.) At least a processing unit (400) to control and communicate with units within the apparatus; characterized in that the optical module (313) of the optical body, has a notch (314) to secure at least one optical filter (315) at a 45° angle for optimum detection of fluorescence. The miniature quantitative polymerase chain reaction apparatus as claimed in claim 1, wherein the thermal cycler unit (200) comprises: at least a sample block (205), a Peltier (206), a heat sink (207) and thermal pad(s) (212) sandwiched between the Peltier and sample block, wherein the sample block contains up to 16 sample wells to contain sample. The miniature quantitative polymerase chain reaction apparatus as claimed in claims 1 & 2, wherein the thermal cycler unit (200) houses a sample block (205) having a heat lid assembly (500), sample tubes (202) and uniformly spaced sample wells (201) at a distance of 3 mm - 6 mm from each other and preferably at a distance of 3 mm - 4 mm measured between any two adjacent sample wells. The miniature quantitative polymerase chain reaction apparatus as claimed in claims 1 to 3, wherein the thermal cycler unit (200) houses a heat lid assembly (500) measuring 106 mm X 48mm X 32mm having at least one metal covering wall (211), a lid (203) and a heater (210) present over at least the top side of the sample wells to prevent condensation. The miniature quantitative polymerase chain reaction apparatus as claimed in claims Ito 4, wherein the thermal cycler unit (200) houses a heater and a heat lid assembly having a metal lid (203) made of Aluminium; and wherein the heater (210) is a polyamide heater. The miniature quantitative polymerase chain reaction apparatus as claimed in claim 1, wherein the thermal cycler unit (200) is made of copper and has equally distanced fins; The miniature quantitative polymerase chain reaction apparatus as claimed in claim 1, wherein the optical body unit (300) comprises at least one optical module (313) havingat least an excitation unit (320) to excite sample, at least a detection unit (322) to detect sample fluorescence post sample excitation; and a mobile shaft (326) to transport the optical module from one well to another. The miniature quantitative polymerase chain reaction apparatus as claimed in claim 1, wherein the optical body unit (300) has at least one gasket ring (323) present at the detection unit (322) and at least one gasket ring (321) present at the excitation unit (320)to prevent any seepage of light. The miniature quantitative polymerase chain reaction apparatus as claimed in claim 1, wherein the optical body unit (300) comprises: at least a detection unit (322), an excitation unit (320), a mobile shaft (326) and at least one optical module (313) having an optical filter fixed within a notch (314) to maintain a 45° position; and wherein the optical filter (315) is sandwiched between a top gasket (318) touching the top block (316) and a bottom gasket (319) sitting within the notch on the bottom block (317). The miniature quantitative polymerase chain reaction apparatus as claimed in claims 1,7, 8 and 9 wherein the optical module has at least one optical filter (315) selected firoma group comprising of a beam splitter optical detection system, a hybrid optical detection system, bifurcated optical detection system, and is preferably a beam splitteroptical detection system. The miniature quantitative polymerase chain reaction apparatus as claimed in claim 1 wherein the optical body has two or more optical modules The miniature quantitative polymerase chain reaction apparatus as claimed in claims 1,7, 8 and 9 wherein the optical module has optical filter(s) maintained at a constant distance from the object lens at any given point. The miniature quantitative polymerase chain reaction apparatus as claimed in claim 1, wherein the processing unit has a PCB operated by a software to at least control temperature of the quantitative polymerase chain reaction apparatus (100) and processing outputs of the detectors for analysis. >