CN117230161A

CN117230161A - Digital PCR kit for detecting TORCH pathogen

Info

Publication number: CN117230161A
Application number: CN202311489875.8A
Authority: CN
Inventors: 祝令香; 邢振存; 杨文军
Original assignee: Xinyi Manufacturing Technology Beijing Co ltd
Current assignee: Xinyi Manufacturing Technology Beijing Co ltd
Priority date: 2023-11-10
Filing date: 2023-11-10
Publication date: 2023-12-15

Abstract

The application provides a digital PCR kit for detecting TORCH pathogens, which is a digital PCR kit for simultaneously detecting 5 TORCH pathogens in one tube, wherein a final reaction system of the digital PCR kit adopts reverse transcription digital PCR, and the kit comprises a primer and probe combination for detecting the TORCH pathogens, and the primer and probe combination is used for simultaneously detecting 5 TORCH pathogens in one tube in a PCR multiplex reaction. The primer and probe combination provided by the application is convenient for rapid screening of TORCH pathogens, and can be accurately quantified, and is simple to operate and short in time consumption.

Description

Digital PCR kit for detecting TORCH pathogen

Technical Field

The application relates to the field of digital PCR detection, in particular to a digital PCR kit for detecting TORCH pathogens.

Background

TORCH refers to a group of pathogens including toxoplasma, rubella virus, cytomegalovirus, herpes simplex virus, other viruses, etc., and pregnant women can infect fetuses or newborns through placenta or birth canal after infection of such pathogens, which may lead to bad prognosis of abortion, premature birth, teratocarcinoma, stillbirth, intrauterine hypoevolutism, neonatal multiple organ damage, etc. For most of the TORCH infections during pregnancy, no effective treatment means is available at present, and it is important to enhance the monitoring of the pre-pregnancy and prenatal TORCH infections.

There are two most common methods for clinical detection of TORCH infection, one for detection of TORCH antibodies and one for detection of TORCH pathogens. Antibodies (IgG, igM) detect an immune response by the body after the virus stimulates the body, are related to the immune function of the individual, and are suitable for screening and immune status assessment. Antibody positivity is an important index for diagnosing active infection, but because the antibody actually detects the immune response state of a human body to TORCH infection, false positives or false negatives are easy to occur for a pregnant woman in a latent state or a pregnant woman with incomplete immune system development. The pathogen (virus antigen, virus DNA/RNA, virus culture) detects the virus itself, and is related to the characteristics of the virus such as replication rule and latent position. In the acute infection stage, blood, amniotic fluid, urine, saliva, herpes fluid, cerebrospinal fluid and other samples are taken, TORCH pathogen culture, antigen detection and nucleic acid detection are carried out, and a positive result is the most direct evidence for judging the acute infection of a specific pathogen. Among them, nucleic acid detection (such as PCR method) is currently the preferred method of clinical pathogen detection. However, the current PCR method is mainly aimed at detecting a single pathogen, and a standard substance and a standard curve are needed to quantitatively analyze the pathogen, so that the operation is complicated. Therefore, there is a need in the clinic for a simple, rapid, accurate quantitative and simultaneous detection of multiple TORCH pathogens and a product and method for early identification of pathogens, early diagnosis by quantitative analysis and rational guidance of clinical medication.

Disclosure of Invention

The application aims to overcome the defect that products for simultaneously and quantitatively detecting 5 TORCH pathogens including toxoplasmosis (Toxoplasma Gondii, TOX), rubella Virus (RV), cytomegalovirus (Cytomegalo.Virus, CMV), herpes simplex Virus I and II (HSV) and parvovirus B19 (B19) are not available at present, and provides a primer and probe combination and a kit for detecting the TORCH pathogens.

In one embodiment, the application provides a digital PCR kit for simultaneous detection of 5 TORCH pathogens in a tube, the digital PCR kit final reaction system employing reverse transcription digital PCR, the kit comprising a primer and probe combination for simultaneous detection of 5 TORCH pathogens in a tube for PCR multiplex reactions, the primer and probe combination being a primer and probe for detection of toxo, rubella virus RV, cytomegalovirus CMV, herpes simplex virus HSV type I and II, and parvovirus B19, the primer and probe being as follows:

wherein m=a or C in CMV-F3, and y=t or C in TOX-F1. Wherein +represents that the base on the right side is locked nucleic acid, and probe IC2-P1 and probe IC2-P2 are probes with the same sequence and marked with different fluorescence.

In one embodiment, the herpes simplex virus I/II probe is a ROX-BHQ2 label, the rubella virus probe is a CY5-BHQ2 label, the toxoplasma probe is a HEX/VIC-BHQ1 label, and the parvovirus probe is a Q705/CY5.5-BHQ3 label, so that 5 TORCH pathogen targets can be detected in one tube by a 5-channel digital PCR instrument, and simultaneously, for an internal standard gene, the detection is performed by using two probes with the same sequence of different labels FAM-BHQ1 and HEX/VIC-BHQ1, so that the fluorescent signal is generated by the internal standard gene in a FAM/VIC double channel, and thus, 6 detection targets can be detected simultaneously by 5 channels according to the position difference of the fluorescent signal groups.

The application aims at the nucleotide sequences of 5 TORCH pathogens, namely toxo, rubella virus RV, cytomegalovirus CMV, herpes simplex virus HSV (I and II) and parvovirus B19, multiple PCR primers and probes were designed for detection of the 5 TORCH pathogens described above by multiple sequence comparison and analysis. Meanwhile, an internal index primer and a probe are also designed and used for monitoring the collection and extraction processes of the sample, so that false negative results are avoided.

The application provides a multiplex PCR primer and probe combination for detecting 5 TORCH pathogens, wherein the primer and probe combination are used for detecting toxoplasmosis TOX, rubella virus RV, cytomegalovirus CMV, herpes simplex virus HSV (I and II) and parvovirus B19, and the multiplex PCR primer probe of the herpes simplex virus HSV designed by the application is a universal primer probe of corresponding viruses, and can detect HSV type I and II.

Because the multiplex PCR primer and probe combination of the application has similar Tm values, hybridization between the primer and probe and the template is facilitated at the same annealing temperature, and the hybridization result is not affected by the temperature problem, so that the detection accuracy is obviously increased. The multiplex PCR primer and probe combination can detect more than 5 TORCH pathogens at one time, has strong detection specificity and accurate detection result, can perform quantitative analysis by a digital PCR method, and does not need a standard curve and a standard substance.

Detailed Description

In order that those skilled in the art will better understand the present application, the following description will proceed with reference being made to illustrative embodiments, only to the extent that they are described in connection with some, but not all embodiments of the present application. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present application are those conventional in the art. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, shall fall within the scope of the application.

Example 1 herpes simplex virus HSV primer probe screening

Herpes simplex virus is one of the viruses that cause skin to appear as herpes simplex. Herpes simplex viruses have 2 serotypes, which are classified as type I and type II. The two types of differences are mainly caused by nucleic acid differences of viruses, and the nucleotide sequences of the viruses have 50% homology, in the application, the nucleotide sequences of HSV type I and type II representative strains are downloaded in NCBI database, primers and probes are designed in 3 common conserved sequence areas of HSV type I and HSV type II through multiple sequence comparison and analysis, 2-3 pairs of primer probes are designed in each conserved region, and the sequences of the primer probes are as follows:

TABLE 1 HSV primer probe sequences

* : + indicates that the base to the right is locked nucleic acid LNA

In HSV single primer probe screening, primer probe screening was performed using DNA amplification mix. The DNA amplification system was prepared as follows:

* Note 1: wherein dU/dNTPs contain dUTP, and the final concentration of dUTP and other 3 dNTPs (dATP, dCTP, dGTP) in a 30 mu L system is 600 mM mM and 200mM respectively. The following is the same as

* Note 2: in a 30 [ mu ] L system, the final concentration of each primer is 600nM, and the final concentration of each probe is 300nM. The following is the same (unless primer probe concentrations are indicated separately).

In screening HSV primer probes, templates used include: 1) HSV type I and type II nucleic acids (from national references of China food and drug inspection institute) were used to evaluate the quantitative accuracy of primer probes using two concentrations (about 200 copies/[ mu ] L and 20 copies/[ mu ] L); 2) Cross-reactive template 1 (including other 4 TORCH pathogen nucleic acids, each pathogen concentration 2000 copies/μl) assessed the specificity of the primer probes; 3) Cross-reactive template 2 (mixed with a number of other pathogen nucleic acids including HIV, EBV, VZV, BKV, e.coli, s.aureus, each pathogen concentration 2000 copies/μl). The nucleic acids in these cross-reaction experiments were derived from national references of the national food and drug verification institute. The primer probe screening criteria were as follows: the high copy and low copy templates of HSV type I and type II were quantified to meet the expected requirements (high copy about 1000 copies/system; low copy number about 80-120 copies/system), and the cross-reactive templates 1 and 2 were not amplified, and the detection signals were FAM channel signals.

The digital PCR workflow is as follows:

(1) Micro-droplet preparation: using a droplet generation chip (new manufacturing technology (Beijing) limited company) and a sample preparation instrument (new manufacturing technology (Beijing) limited company), adding a 30 mu L PCR reaction system into a sample hole of the droplet generation chip, adding 160 mu L droplet generation oil into the oil hole, placing the chip and 8 row pipes into the preparation instrument, covering a rubber mat, and performing micro droplet preparation.

(2) And (3) PCR amplification: the 8-row tube containing the microdroplets was placed on a PCR instrument for amplification, and the amplification procedure was set as shown in the following table:

(3) And (3) detecting micro liquid drops: after the PCR is completed, the 8-row tube and a liquid drop detection chip (New manufacturing technology (Beijing) limited company) are placed in a fixture, 430 mu L of detection oil and 500 mu L of detection oil are respectively added into the oil holes, a rubber mat is covered, and the chip is placed in a chip analyzer (New manufacturing technology (Beijing) limited company) for liquid drop detection.

(4) Data analysis: after PCR amplification, each micro-droplet is detected by a chip analyzer, the fluorescence signal intensity of the micro-droplet signal is recorded, and the droplet containing the target gene to be detected is detected to obtain a corresponding fluorescence signal. The fluorescence intensity in the micro-droplets is digitized through a fluorescence classification threshold value, the micro-droplets with stronger fluorescence are judged to be 1 (positive), the micro-droplets with weaker fluorescence are judged to be 0 (negative), the numbers of 1 and 0 are counted, and the total copy number of the target genes put into the template can be calculated through poisson distribution model correction.

The HSV single primer probe screening results are shown in table 2 below. According to the set interpretation criteria, 3 sets of qualified primers and probes (HSV-F5/R5/P5, HSV-F6/R6/P6, HSV-F7/R7/P7) were screened for subsequent evaluation and multiplex reaction combinations. In the application, primers and probes are designed in 3 common conserved sequence regions of HSV type I and HSV type II, 3 pairs of primers and probes designed in a herpes simplex virus I/II conserved region 1 are unsuitable, 2 pairs of primers and probes designed in a herpes simplex virus I/II conserved region 2 are only available, and 3 pairs of primers and probes designed in a herpes simplex virus I/II conserved region 3 are only available. Therefore, when the herpes simplex virus I/II is detected simultaneously, the detection of the herpes simplex virus I/II can be realized simultaneously by further screening specific conserved regions and designing specific primers and probes on the basis of determining the common conserved regions.

TABLE 2 screening results of HSV primer probes

。

Example 2 three DNA pathogen (CMV, TOX and parvovirus B19) primer probe screening

Three pathogens including cytomegalovirus CMV, toxoplasma gondii TOX and parvovirus B19 representative strains were downloaded in NCBI database, primers and probes were designed in a common conserved region of each pathogen representative strain sequence by multiple sequence comparison and analysis, 2-3 pairs of primer probes were designed for each pathogen and the primer probe sequences were as follows:

TABLE 3 CMV, TOX and parvoviral B19 primer probe sequences

* : + indicates that the base to the right is locked nucleic acid LNA

Primer probe screening was performed using DNA amplification mix. The DNA amplification system was formulated as in example 1.

In screening CMV, TOX and parvovirus B19 primer probes, templates used include: 1) CMV, TOX and parvovirus B19 nucleic acids (from national references of the chinese food and drug verification institute) were used to evaluate the quantitative accuracy of primer probes using two concentrations (about 200 copies/μl and 20 copies/μl); 2) Cross-reactive template 1 (including other 4 TORCH pathogen nucleic acids except for the detection target, 2000 copies per μl pathogen concentration) evaluates the specificity of the primer probes; 3) Cross-reactive template 2 (mixed with a number of other pathogen nucleic acids including HIV, EBV, VZV, BKV, e.coli, s.aureus, each pathogen concentration 2000 copies/μl). The nucleic acids in these cross-reaction experiments were derived from national references of the national food and drug verification institute. The primer probe screening criteria were as follows: the high copy and low copy template quantification for CMV, TOX and parvovirus B19 targets met the expected requirements (high copy about 1000 copies/system; low copy number about 80-120 copies/system) and no amplification was performed for cross-reactive templates 1 and 2, and the detection signals were FAM channel signals. The digital PCR workflow is as described in example 1.

CMV, TOX and B19 single primer probe screening results are shown in tables 4-6 below. According to the set interpretation criteria, 6 sets of qualified primers and probes (CMV-F1/R2/P1, CMV-F3/R3/P2, TOX-F1/R1/P1, TOX-F2/R2/P1, B19-F1/R1/P1, B19-F2/R2/P1) were screened for subsequent evaluation and multiplex reaction combinations.

TABLE 4 CMV primer probe screening results

TABLE 5 TOX primer probe screening results

。

TABLE 6B 19 primer probe screening results

。

Example 3 rubella virus RV primer probe screening

Rubella virus is RNA virus, nucleotide sequences of rubella virus RV representative strains are downloaded in NCBI database, and primers and probes are designed in a common conserved region of pathogen representative strain sequences through multiple sequence comparison and analysis, wherein the sequences of the primers and probes are as follows:

TABLE 7 RV primer probe sequences

* : + indicates that the base to the right is locked nucleic acid LNA

Primer probe screening was performed using reverse transcription amplification mix. The reverse transcription amplification system was formulated as follows:

* Note 1: wherein dU/dNTPs contain dUTP, and the final concentration of dUTP and other 3 dNTPs (dATP, dCTP, dGTP) in a 30 mu L system is 600 mM mM and 200mM respectively.

* Note 2: in a 30 [ mu ] L system, the final concentration of each primer is 600nM, and the final concentration of each probe is 300nM.

In screening RV primer probes, templates used include: 1) Rubella virus nucleic acid (from national reference of China food and drug inspection institute) is used for evaluating the quantitative accuracy of the primer probe by adopting two concentrations (about 200 copies/mu L and 20 copies/mu L); 2) Cross-reactive template 1 (including other 4 TORCH pathogen nucleic acids except RV, 2000 copies/μl per pathogen concentration) assessed the specificity of the primer probes; 3) Cross-reactive template 2 (mixed with a number of other pathogen nucleic acids including HIV, EBV, VZV, BKV, e.coli, s.aureus, each pathogen concentration 2000 copies/μl). The nucleic acids in these cross-reaction experiments were derived from national references of the national food and drug verification institute. The primer probe screening criteria were as follows: the high copy and low copy template quantification of RV targets meets the expected requirements (high copy about 1000 copies/system; low copy number about 80-120 copies/system) and no amplification of cross-reactive templates 1 and 2, both detection signals were FAM channel signals.

The digital PCR workflow is as described in example 1. Wherein a reverse transcription step is added before the PCR amplification step, and the RNA reverse transcription procedure is as follows: reverse transcription is carried out at 55℃for 15 min, followed by PCR amplification as described in example 1.

RV single primer probe screening results are shown in Table 8 below. Based on the set criteria, 1 set of qualified primers and probes (RV-F1/R1/P1) were screened together for subsequent evaluation and multiplex reaction combinations.

TABLE 8 RV primer probe screening results

EXAMPLE 4 reverse transcription System specific selection primer probe combinations

The object of the present application is to detect 5 TORCH pathogens by a one-tube system, thus requiring the use of human genome total nucleic acid and cross-templates as templates for detection of non-specific reverse transcription and amplification, and eliminating the occurrence of non-specific signals due to non-specific reverse transcription and amplification.

When a reverse transcription system is used for screening primer probe combinations, the template comprises: 1) TORCH target nucleic acid (from national reference of the national institute of food and drug verification), high concentration (about 200 copies/μl) was used to evaluate the quantitative accuracy of primer probes; 2) Cross-reactive template 2 (mixed with a plurality of other pathogen nucleic acids, including HIV, EBV, VZV, BKV, e.coli, s.aureus, each pathogen concentration 2000 copies/μl, nucleic acids derived from national references of the national food and drug verification institute); 3) Human genomic nucleic acid (100 ng/μl, including DNA and RNA). The criteria for primer probe combination screening were as follows: the high copy template quantification for each target meets the expected requirements (about 1000 copies/system) and the detection signals are FAM channel signals without amplification of cross-reactive template 2 and human genomic nucleic acid.

The reverse transcription specific screening results are shown in Table 9 below. According to the set interpretation standard, 8 groups of qualified primers and probes (HSV-F5/R5/P5, CMV-F1/R2/P1, CMV-F3/R3/P2, TOX-F1/R1/P1, TOX-F2/R2/P1, B19-F1/R1/P1, B19-F2/R2/P1, RV-F1/R1/P1) are screened out for multiple reaction combinations.

TABLE 9 reverse transcription specificity screening results

EXAMPLE 5 TORCH multiplex amplification System screening

The qualified primer probes in Table 9 above were combined into a multiplex reverse transcription digital PCR amplification system, and the amplification system was screened for detection of 5 TORCH pathogens. Wherein, the primer probes qualified by CMV, TOX, B are respectively provided with two groups (CMV-F1/R2/P1, CMV-F3/R3/P2, TOX-F1/R1/P1, TOX-F2/R2/P1, B19-F1/R1/P1 and B19-F2/R2/P1), and the primer probes qualified by HSV and RV are respectively provided with 1 group (HSV-F5/R5/P5 and RV-F1/R1/P1), so that the combination is 8 combinations in a tube 5 weight system. The amplification system was a reverse transcription digital PCR system formulated as in example 3 and the digital PCR procedure was as described in example 1. Wherein the final concentration of each primer in the 20-primer probe is 600nM and the final concentration of the probe is 300nM. The amplification procedure is also described for reverse transcription and PCR amplification of RNA in example 3.

The templates used included: 1) TOX, CMV, HSVI and II, B19 pathogen nucleic acids (both from national references of the national institute of food and drug) were used at two concentrations (about 200 copies/μl and 20 copies/μl); 2) Cross-reactive template 2 (mixed with a plurality of other pathogen nucleic acids, including HIV, EBV, VZV, BKV, e.coli, s.aureus, each pathogen concentration 2000 copies/μl, nucleic acids derived from national references of the national food and drug verification institute); 3) Human genomic nucleic acid (100 ng/μl, including DNA and RNA). The criteria for screening the multiple reaction system are as follows: high copy and low copy template quantification for 5 pathogens meets expected requirements (high copy about 1000 copies/system; low copy number about 80-120 copies/system), no amplification for cross-reactive templates; no non-specific amplification is carried out on human genome, and detection signals are FAM channels.

The test results are shown in Table 10. The results show that, in combination 2: CMV-F3/R3/P2; HSV-F5/R5/P5; TOX-F1/R1/P1; B19-F1/R1/P1; the detection results of RV-F1/R1/P1 on 5 pathogens, cross templates and human genome nucleic acid all meet the expectations, and the primer probe combination is used for screening internal standard genes as the next step.

EXAMPLE 6 screening of internal reference Gene

And adding internal standard genes into a multiple TORCH detection system for quality control of the whole extraction-detection process. Two sets of primers and probes for internal standard genes, labeled HEX-BHQ1 as shown in Table 11, were designed, and multiplex amplification was performed together with the multiplex primer probe combination 2 selected in example 5 to screen for a suitable internal standard gene.

TABLE 11 internal reference primer probe sequences

The amplification system was a reverse transcription digital PCR system formulated as in example 3 and the digital PCR procedure was as described in example 1. Wherein the final concentration of each primer in the 20-primer probe is 600nM and the final concentration of the probe is 300nM. The amplification procedure is also described for reverse transcription and PCR amplification of RNA in example 3.

The templates used included: 1) TOX, CMV, HSVI and II, B19 pathogen nucleic acids (both from national references of the national institute of food and drug) were used at two concentrations (about 200 copies/μl and 20 copies/μl); 2) Cross-reactive template 2 (mixed with a plurality of other pathogen nucleic acids, including HIV, EBV, VZV, BKV, e.coli, s.aureus, each pathogen concentration 2000 copies/μl, nucleic acids derived from national references of the national food and drug verification institute); 3) Human genomic nucleic acid (10 ng/μl, including DNA and RNA). The screening criteria for the multiple reaction system containing the internal standard gene are as follows: high copy and low copy template quantification for 5 pathogens meets expected requirements (high copy about 1000 copies/system; low copy number about 80-120 copies/system), no amplification for cross-reactive templates; the internal standard gene IC (VIC channel) copy number was about 3000 copies, and the target primer probe (FAM channel) did not have non-specific amplification on human genomic samples.

The test results are shown in Table 12. The results show that the primer probe of the internal standard gene 2 and the combined 2 primer probe are combined together, and the detection results of 5 pathogens, cross templates and human genome nucleic acid are all expected.

TABLE 12 screening results of internal Standard Gene primer probes

EXAMPLE 7 multiple digital PCR TORCH detection System typing

Since multiple reactions were performed in one tube, the fluorescent label was modified for a portion of the probes, wherein the HSV probe was modified to a ROX-BHQ2 label, the rubella virus probe was modified to a CY5-BHQ2 label, the toxoplasma was modified to a HEX/VIC-BHQ1 label, and the parvovirus B19 was modified to a Q705/CY5.5-BHQ3 label, to achieve detection of 5 TORCH pathogen targets in one tube by a 5-channel digital PCR instrument while for the internal reference gene, detection was performed with probes of the same sequence of two different labels (FAM-BHQ 1 and HEX/VIC-BHQ 1), to achieve fluorescent signal generation for the internal reference gene for FAM/VIC double channels, so that 6 detection targets could be simultaneously detected by 5 channels based on the positional difference of the fluorescent signal clusters. The final primer probe sequences are shown in Table 13.

TABLE 13 finally determined primer probe sequences

Using the primer probe formulation system in Table 13, the amplification system was a reverse transcription digital PCR system, the system formulation was as described in example 3 and the digital PCR procedure was as described in example 1. Wherein the final concentration of each primer of TORCH 5 targets in the 20-point primer probe is 600nM and the final concentration of the probe is 300nM; the final primer concentration of the internal standard gene was 400nM, the final FAM probe concentration was 200nM, and the final HEX probe concentration was 150nM. The amplification procedure is also described for reverse transcription and PCR amplification of RNA in example 3.

The templates used included: 1) TOX, CMV, HSVI and II, B19 pathogen nucleic acids (both from national references of the national institute of food and drug) were used at two concentrations (about 200 copies/μl and 20 copies/μl); 2) Cross-reactive template 2 (mixed with a plurality of other pathogen nucleic acids, including HIV, EBV, VZV, BKV, e.coli, s.aureus, each pathogen concentration 2000 copies/μl, nucleic acids derived from national references of the national food and drug verification institute); 3) Human genomic nucleic acid (10 ng/μl, including DNA and RNA); 4) TOX, CMV, HSVI and II, B19, RV viral nucleic acid and human genomic nucleic acid were mixed into multiplex templates, comprising two concentrations (about 200 copies/μl and 20 copies/μl per TORCH pathogen target, internal standard gene about 3000 copies/system and 300 copies/system). The multiple reaction system determination criteria are as follows: high copy and low copy template quantification for 5 single pathogens meets expected requirements (high copy about 1000 copies/system; low copy number about 80-120 copies/system), quantification for human genome about 3000 copies/system, no amplification for cross-reactive templates; multiple templates need to be detected simultaneously and the quantification meets the requirements (about 1000 copies per target at high copy per system; about 80-120 copies per target at low copy per system).

The test results are shown in Table 14. The result shows that the constructed multiple digital PCR TORCH detection system can meet the set judgment standard, can quantitatively detect the single template and the multiple templates, has no cross non-specific reaction and is shaped.

TABLE 14 multiplex assay results

Example 8 TORCH multiplex RT-dPCR detection kit

Based on the multiplex PCR primers and probes described in example 7, the present application developed a multiplex RT-dPCR detection kit for detecting the 5 TORCH pathogens described above. The kit contains primer probes shown in Table 13 as SEQ ID NOs: 1-19, a 20-point primer probe detection solution, a PCR reagent A (containing dPCR buffer solution, dUTP/dNTPs, mgCl2 and DNA polymerase), a PCR reagent B (reverse transcriptase and RNase inhibitor), a positive reference substance and DEPC water.

The kit is provided with an internal standard gene system for monitoring the sample collecting and extracting processes and avoiding false negative results. The PCR detection system comprises UNG enzyme and dUTP pollution prevention measures, and can fully degrade the pollution of the possibly existing PCR products and avoid the false positive result.

Example 9 RT-dPCR detection method of TORCH disease pathogen

The present application is exemplified by the kit of example 8, and a method for detecting the 5 TORCH pathogens for the purpose of non-disease diagnosis using the kit is briefly described.

The method comprises the following steps: (1) Extracting or releasing nucleic acid of the sample to be tested by using a commercial kit;

(2) RT-dPCR amplification;

using the primers and probes in the kit SEQ ID NO: 1-19, carrying out RT-dPCR amplification on the sample to be detected, wherein the sample loading amount of the nucleic acid is 5 mu L, and the total reaction volume is 30 mu L. The reaction system for PCR amplification is shown in Table 15:

TABLE 15 multiplex PCR reaction System

* Note 2: in a 30 [ mu ] L system, the final concentration of the target index primer is 600nM, the final concentration of the target probe is 300nM, the concentration of the internal standard gene primer is 400nM, and the final concentrations of the two probes are 200nM and 150nM respectively.

The application can amplify 5 TORCH pathogens and internal standard genes under the same condition, reduces the dosage of PCR reagents and the demand of instruments, reduces operation steps and reduces cost.

Example 10 Linear Range and minimum detection limit test

The test was performed using plasmids for the target sequences of toxoplasma, cytomegalovirus, herpes simplex virus and parvovirus B19, and rubella virus pseudoviruses (all from general biosystems (Anhui), inc.), for the linear range of detection and sensitivity (lowest detection limit) of the primer and probe combinations of the application (combination 2+ internal standard gene 2).

Accuracy and linear correlation assessment

(1) Nucleic acid extraction

5 plasmids to be tested and pseudoviruses were mixed in a negative sample as artificial simulated samples, and DNA and RNA were extracted using a nucleic acid extraction kit, and specific steps were performed with reference to the New manufacturing technology (Beijing) Limited nucleic acid extraction kit instructions.

(2) Nucleic acid dilution

The above-mentioned extracted sample nucleic acids were subjected to multiple dilution, respectively, so that the detection concentrations were 12500 copies/reaction, 2500 copies/reaction, 500 copies/reaction, 100 copies/reaction, 20 copies/reaction, 5 copies/reaction in this order.

(3) RT-PCR amplification

RT-dPCR amplification and detection were performed with reference to the reaction system described in Table 15 and the following reaction procedure.

The RNA reverse transcription procedure was: reverse transcription is carried out at 55 ℃ for 15 min.

The PCR amplification procedure was as follows:

(4) Interpretation of results

And judging the result according to the set threshold value of the positive judgment value (the threshold value of the target and the internal standard gene is 3 copies/system). When the copy number of the target gene is greater than or equal to a threshold value, the sample is positive to the target; when the copy number of the target gene is smaller than the threshold value and the copy number of the internal standard gene is larger than the threshold value, the sample is negative to the target; when the target gene copy number is less than the threshold and the internal standard gene copy number is less than the threshold, the sample is undetected, and inhibition of the template or too low a nucleic acid content is possible, and extraction and detection are recommended again.

The linear range test results after gradient dilution of 5 simulated samples are shown in table 16. As shown in the results of Table 16, the primer and the probe of the application are used for detecting 5 artificial simulation samples, the linear correlation and the accuracy are good, the linear correlation R2 is more than 0.99, the samples can be absolutely quantified, and the quantitative accuracy is good. From the results of the nucleic acid dilution detection concentration, the minimum detection limit was about 5 copies/reaction.

TABLE 16 Linear Range detection results

(two) sensitivity and minimum detection limit test

The application detects the samples of the 5 artificial simulation samples which are subjected to gradient dilution to the concentration near the detection limit (10 copies/system, 5 copies/system and 2.5 copies/system), each sample is detected for 10 times, the result is shown in the following table 17, each target is specifically detected, under the condition of the target copy number of the 5 copies/system, the positive rate of 10 times repeated detection of each detection target is 10/10, and the detection sensitivity of 5 TORCH pathogens detected by the primer and the probe is 5 copies/system, the sensitivity is high, and the detection result is accurate.

TABLE 17 minimum detection limit results

Example 11 test of Mixed detection Capacity of kit

The application utilizes the kit described in the embodiment 8 to detect the mixed artificial simulation sample of different pathogen nucleic acids so as to test the mixed detection effect. In this example, a mixed sample of HSV type I/CMV, B19/RV, TOX/CMV (500 copies/system per target concentration) was taken as an example to demonstrate the effect of the mixing test, and the results are shown in Table 18 below. The result shows that the detection result of the kit for detecting the mixed sample is normal, and the non-specific result does not appear, so that the kit can be used for detecting different viruses at the same time, and the quantitative result of the mixed sample is accurate.

TABLE 18 template Mixed inspection results

EXAMPLE 12 detection of different HSV genotypes

The HSV primer probe designed by the application is a universal primer probe, and can detect HSV type I and type II virus nucleic acid simultaneously. The two types of viral nucleic acids were tested using the kit described in example 8 and the test results are shown in table 19 below. The results show that the kit can accurately detect the type I HSV and the type II HSV and can be used for identification verification and quantitative analysis of the parting viruses.

TABLE 19 detection results of different HSV types

It is to be understood that this application is not limited to the particular methodology, protocols, and materials described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present application which will be limited only by the appended claims.

Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the application described herein. Such equivalents are also encompassed by the appended claims.

Claims

1. A digital PCR kit for detecting TORCH pathogens, wherein the kit is a digital PCR kit for simultaneously detecting 5 TORCH pathogens in a tube, the digital PCR kit final reaction system employs reverse transcription digital PCR, the kit comprises a primer and probe combination for detecting TORCH pathogens, the primer and probe combination is a primer and probe needle for detecting toxoplasma torae TOX, rubella virus RV, cytomegalovirus CMV, herpes simplex virus HSV type I and II, and parvovirus B19 simultaneously in a tube, the primer and probe combination is as follows:

；

wherein m=a or C in CMV-F3, TOX-F1 is y=t or C; wherein +represents that the base on the right side is locked nucleic acid, and probe IC2-P1 and probe IC2-P2 are probes with the same sequence and marked with different fluorescence.

2. The digital PCR kit according to claim 1, wherein the herpes simplex virus I/II probe is a ROX-BHQ2 label, the rubella virus probe is a CY5-BHQ2 label, the toxoplasma probe is a HEX/VIC-BHQ1 label, and the parvovirus probe is a Q705/CY5.5-BHQ3 label, to enable detection of 5 TORCH pathogen targets in one tube by a 5-channel digital PCR instrument, while for the internal reference gene, detection is performed with two differently labeled probes of the same sequence as the FAM-BHQ1 and HEX/VIC-BHQ1, to enable generation of fluorescent signals for the internal reference gene as FAM/VIC dual channels, such that 6 detection targets are simultaneously detected by 5 channels based on the difference in the location of the fluorescent signal groups.