CN112725527A - SARS-CoV-2 full premixed freeze drying fluorescence PCR detection kit and method thereof - Google Patents

SARS-CoV-2 full premixed freeze drying fluorescence PCR detection kit and method thereof Download PDF

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CN112725527A
CN112725527A CN202011571674.9A CN202011571674A CN112725527A CN 112725527 A CN112725527 A CN 112725527A CN 202011571674 A CN202011571674 A CN 202011571674A CN 112725527 A CN112725527 A CN 112725527A
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freeze
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sars
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李然栋
马清霞
薄慧文
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Qingdao Batfi Technology Development Co ltd
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Qingdao Batfi Technology Development Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes

Abstract

The invention discloses a SARS-CoV-2 full premix freeze-drying fluorescence PCR detection kit, which comprises a freeze-drying solid SARS-CoV-2RT-PCRMix, a liquid redissolving Buffer, a freeze-drying solid positive control and a freeze-drying solid negative control, wherein the freeze-drying solid SARS-CoV-2RT-PCRMix contains a primer group corresponding to primer sequences of SARS-CoV-2 specific gene ORF1ab, N gene and human source internal reference gene RNAseP. The method is simple and convenient to operate, has low requirement on the operation level of detection personnel, and can realize quick screening of large samples with low cost. The kit only needs 40 minutes to 1 hour for whole detection, and the result is accurate and reliable.

Description

SARS-CoV-2 full premixed freeze drying fluorescence PCR detection kit and method thereof
Technical Field
The invention belongs to the field of virus rapid detection reagents, and particularly relates to a SARS-CoV-2 full-premix freeze-drying fluorescence PCR detection kit and a method thereof.
Background
2019 coronavirus Disease (Corona Virus Disease 2019, COVID-19) is an acute respiratory infectious Disease caused by a novel coronavirus (New Corona Virus, SARS-CoV-2). SARS-CoV-2 belongs to a novel coronavirus of the genus beta, which is an RNA virus, which is enveloped, has a circular or elliptical particle, is usually polymorphic, has a diameter of 60-140nm, is the 7 th coronavirus which is known to infect humans at present, and the other 6 coronaviruses are HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV, respectively. The first 4 of these are more common in the population, less pathogenic, and generally cause only mild respiratory symptoms like the common cold, while the other 2 are the more pathogenic SARS coronavirus and MERS coronavirus. Nucleic acid detection is a common method for clinical etiology diagnosis, and has the advantages of high sensitivity, strong specificity and the like. At present, the real-time fluorescence RT-PCR technology is listed in the national SARS-CoV-2 diagnosis and treatment guideline, and all national county level disease control centers require the development of fluorescence RT-PCR detection. However, the fluorescent RT-PCR detection technology has high requirements on the technical level of laboratory hardware and technicians, and the detection results are difficult to achieve accuracy and consistency due to the fact that the hardware and the technicians of the PCR detection rooms in the national disease control centers are different.
Therefore, it is urgently needed to develop a detection kit with high stability and simple operation, which reduces the influence of repeated freezing and thawing of reagents on a PCR system in the transportation and storage processes, reduces the influence of unskilled operation of laboratory personnel on a detection result, and improves the stability, the repeatability and the uniformity of a fluorescence RT-PCR detection result.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a SARS-CoV-2 full-premix freeze-drying fluorescence PCR detection kit, which comprises a freeze-drying solid SARS-CoV-2RT-PCR Mix, a liquid redissolving Buffer, a freeze-drying solid positive control and a freeze-drying solid negative control, wherein the freeze-drying solid SARS-CoV-2RT-PCR Mix contains a primer group corresponding to primer sequences of a SARS-CoV-2 specific gene ORF1ab, an N gene and a human internal reference gene RNAse P.
Further, the primer group sequences corresponding to the SARS-CoV-2 specific ORF1ab gene and the N gene are as follows:
ORF1ab-F：AAAGGTTATGGCTGTAGTTG
ORF1ab-R：CGATTGTGCATCAGCTGACTG
ORF1ab-P：TCAACTCCGCGAACCCATGCTT
n gene-F: GTCAAGCCTCTTCTCGTTCC
N gene-R: GGAGAAGTTCCCCTACTGC
N gene-P: TTGAACTGTTGCGACTACGTGAT
The primer group corresponding to the primer sequence of the human reference gene RNAse P is as follows:
RNAse P-F：AGATTTGGACCTGCGAGCG
RNAse P-R：GAGCGGCTGTCTCCACAAGT
RNAse P-P：TTCTGACCTGAAGGCTCTGCGCG。
furthermore, the freeze-dried solid SARS-CoV-2RT-PCR Mix contains 0.1-0.8 mmol/L dNTP, 0.01-0.1U/muL Taq DNA polymerase, 1-10U/muL MLV reverse transcriptase, 0.1-2U/muL RNase inhibitor, 0.01-0.06U/muL LUDG enzyme, 0.2-0.9 mumol/L ORF1ab, 0.2-0.9 mumol/L N gene, mumol/L primer of RNase P, probe, 3-6% trehalose, 0.01-0.1% glycine, DTT and 0.1-1% bovine serum albumin before freeze-drying, and the freeze-drying protective agent is prepared after mixing.
Further, the complex solution contains 3-10 mmol/LMgSO4, 7-13 mmol/LKCl, 13-22 mmol/L Tris-HCl, 8-15 mmol/L (NH4)2SO4, 0.1-0.3% Triton X-100 and 0.3-1.6 mol/L betaine.
Furthermore, the positive control is lyophilized solid RNA corresponding to SARS-CoV-2 specific gene ORF1ab and N synthesized in vitro, and the negative control is purified non-SARS-CoV-2 artificially synthesized lyophilized solid RNA.
The invention also provides a SARS-CoV-2 full premix freeze-drying fluorescence PCR detection reagent method, which comprises the following steps:
(1) preparing a reagent: adding 15 mu L of redissolving Buffer RB into each PCR tube with the freeze-dried particles, and adding 50ul of redissolving Buffer RB into freeze-dried penicillin bottles of positive control and negative control;
(2) sample and control addition: adding 5ul of extracted sample RNA into each PCR tube according to the number of experimental samples, adding 5ul of redissolved negative and positive controls into the corresponding PCR tube, and covering the PCR tube;
(3) centrifuging: and (3) centrifuging the PCR tube for a short time, and then putting the tube into a fluorescent quantitative PCR instrument.
(4) Setting PCR parameters: simultaneously selecting FAM, HEX and Cy5 channels from the instrument channel to respectively correspond to ORF1ab, N gene and internal reference RNAse P genes;
(5) and (4) carrying out PCR result analysis, verifying whether the test is established and judging the result.
Further, after the PCR parameters are set in the step (4), the PCR amplification procedure is carried out for 15min at 50 ℃ and 3min at 95 ℃ in a circulating manner once; the fluorescence was collected at 60 ℃ after 45 cycles of 95 ℃ for 15s and 60 ℃ for 60 s.
Further, the specific step of determining the result in step (5) is to establish a test when the negative control has no expansion curve in both FAM and HEX channels and the CT value of the positive control in both FAM and HEX channels is < 38. When the CT value of the sample is less than or equal to 40 and the sample has a typical S-shaped amplification curve, the sample is judged to be positive; when the CT value of the sample is greater than 40 and no typical S-shaped curve exists, the sample is judged to be negative.
Compared with the prior art, the invention has the beneficial effects that: the kit is used for rapidly detecting SARS-CoV-2 based on the fluorescent quantitative RT-PCR technology, uses 2 pairs of special primers and human internal reference genes, specifically amplifies a specific sequence in vitro, and combines a fluorescent probe for real-time detection. The method is simple and convenient to operate, has low requirement on the operation level of detection personnel, and can realize quick screening of large samples with low cost. The kit only needs 45 minutes to 1 hour for whole-process detection, has accurate and reliable result, and is specifically represented as follows:
1. the fluorescent quantitative PCR detection reagent combines the freeze-drying technology, and after the composite freeze-drying protective agent is added and the freeze-drying process is optimized, the stability of the reagent is greatly improved, the normal-temperature transportation is convenient, and the transportation cost is saved;
2. the SARS-CoV-2RT-PCRMix freeze-drying reagent mixes the components of a primer, a probe, a reverse transcriptase, a PCR reaction enzyme and a PCR buffer system of RT-PCR reaction uniformly according to an optimized proportion and subpackages the mixture into a PCR tube, the reagent can be used after dissolution, repeated freeze thawing and repeated subpackaging are avoided, the use is simple, convenient and quick, and the reagent is more suitable for basic-level field detection;
3. meanwhile, the human-derived reference gene is detected, so that the quality of a clinical sample can be monitored, and the reliability of a detection result is improved.
4. The amount of the template required by the reaction is small, the sensitivity is high, and the lowest detection limit can be as low as 10 copy/Test;
5. the UDG enzyme is added into the reaction system, so that the false positive result caused by aerosol pollution can be effectively reduced;
6. 2 pairs of special primers for specifically detecting the target gene sequence are utilized to specifically recognize 6 independent regions on the target sequence, the high specificity is realized, the whole PCR reaction is completed within 45min, and the detection result is displayed in real time.
Drawings
FIG. 1 is the amplification curve diagram of SARS-CoV full premix freeze dry PCR analysis sensitivity test.
FIG. 2 is a graph showing the amplification curve of SARS-CoV full premix freeze dry PCR analysis specificity test.
Detailed Description
The technical solution of the present invention will be described in further detail with reference to the accompanying drawings and the detailed description.
Example 1
A freeze-dried solid SARS-CoV-2 rapid detection reagent capable of being transported and stored at normal temperature. The kit is pre-installed in a PCR reaction tube, and testers only need to extract nucleic acid from a sample and add the nucleic acid into the PCR reaction tube, so that the whole operation can be completed, the kit is convenient and quick, and the requirement of the current epidemic situation on the rapid detection of SARS-CoV-2 can be met.
The quick detection kit for detecting SARS-CoV-2 includes solid SARS-CoV-2RT-PCR Mix, liquid redissolving Buffer, solid positive control and solid negative control. The freeze-dried solid SARS-CoV-2RT-PCR Mix detection reagent contains SARS-CoV-2 specific gene ORF1ab, N gene and primer group corresponding to primer sequence of human source internal reference gene RNAse P, and the primer group is SARS-CoV-2 specific ORF1ab gene and N gene.
The primer sequences corresponding to the SARS-CoV-2 specific ORF1ab gene and the N gene are as follows:
ORF1ab-F：AAAGGTTATGGCTGTAGTTG
ORF1ab-R：CGATTGTGCATCAGCTGACTG
ORF1ab-P：TCAACTCCGCGAACCCATGCTT
n gene-F: GTCAAGCCTCTTCTCGTTCC
N gene-R: GGAGAAGTTCCCCTACTGC
N gene-P: TTGAACTGTTGCGACTACGTGAT
The primer sequence of the human internal reference gene RNAse P is as follows:
RNAse P-F：AGATTTGGACCTGCGAGCG
RNAse P-R：GAGCGGCTGTCTCCACAAGT
RNAse P-P：TTCTGACCTGAAGGCTCTGCGCG
the freeze-dried solid SARS-CoV-2RT-PCR Mix contains 0.1-0.8 mmol/L dNTP, 0.01-0.1U/mu LTaq DNA polymerase, 1-10U/mu LMMLV reverse transcriptase, 0.1-2U/mu L RNase inhibitor, 0.01-0.06U/mu LUDG enzyme, 0.2-0.9 mu mol/L primer and probe of ORF/N gene/RNase P gene, 3-6% trehalose (w/v), 0.01-0.1% glycine (w/v), 0.1-1% albumin (w/v) bovine serum freeze-dried composite protective agent, and the balance of PCR grade water before freeze-drying.
Preparation of the freeze-dried solid SARS-CoV-2RT-PCRMix
1) Primer synthesis: synthesizing oligonucleotide primers according to primer sequences of ORF1ab gene, N gene and RNase P, and quantitatively preparing;
2) preparing a SARS-CoV-2RT-PCR Mix liquid: mixing the above components under sterile environment.
3) Subpackaging: and subpackaging the product into 8 rows of PCR tubes by using a trace quick dispenser.
4) Half-covered PCR cover: the cover of the PCR tube is half-buckled on the PCR tube without fastening and compacting.
5) Vacuum freeze drying: pre-freezing for 3 hours at-35 ℃ in a freeze dryer. The pressure in the freeze dryer is reduced to below 10Pa and the vacuum treatment is carried out for 2 hours at the temperature of-35 ℃. The temperature inside the freeze dryer was raised to 10 ℃ under vacuum and vacuum-treated for 2 hours. The temperature inside the freeze dryer was raised to 30 ℃ under vacuum for 2 hours.
6) And (4) capping: and clicking an automatic button to execute capping operation after the freezing process is finished.
7) And (3) vacuum packaging: and packaging the capped product on a vacuum packaging machine by using an aluminum foil bag.
Preparing the liquid redissolving Buffer:
the composite solution contains 3-10 mmol/L MgSO4, 7-13 mmol/L KCl, 13-22 mmol/L Tris-HCl, 8-15 mmol/L (NH4)2SO4, 0.1-0.3% Triton X-100 and 0.3-1.6 mol/L betaine. Mixing the above components in sterile environment, and placing in a container; subpackaging: and subpackaging the product into penicillin bottles by using a trace quick dispenser. And (4) capping: and (4) buckling the bottle cover on the penicillin bottle, and clicking an automatic button to perform capping operation.
Preparation of the positive solid lyophilized RNA positive control:
the positive control is SARS-CoV-2 specific gene ORF1ab synthesized in vitro and RNA of N gene fragment.
Preparation of solid-state positive control RNA:
1) in vitro synthesis of positive RNA fragments, first of all the primers T7 CDC F1 and T7 CDC R1, T7 CDC F1: CGACTCACTATAGGGAGATATCGGATCC, T7 CDC R1: TCGTTGTTGGCCTTTACCAGACATTTTG, with the sequence of the chinese CDC positive control plasmid 1: 108After dilution, the PCR product was used as a template for the first PCR amplification. After identification as a specific product, the 1 st PCR product 1: 105Double dilution, with primer pair 2T 7 CDC F2 and T7 CDC R1, T7 CDC F2: CTTAGGTAATACGACTCACTATAGGGA, respectively; t7 CDC R1:
TCGTTGTTGGCCTTTACCAGACATTTTG, performing a second PCR amplification, and purifying the PCR product of the 2 nd amplification with a column nucleic acid extraction kit. Then, using T7 RNA polymerase in vitro reverse transcription kit, the PCR product 1 after 2 nd amplification purification: 103Diluting and carrying out reverse transcription. After the reaction is finished, surplus DNA is removed by using DNase digestion, the column type nucleic acid extraction kit purifies a reverse transcription product, and the obtained purified RNA is the RNA containing SARS-CoV-2 specific gene ORF1ab and N gene fragments.
2) And (3) calibrating the concentration of the positive control RNA, namely determining that the concentration of the positive control RNA is 20-30 ng/mu L, and according to the characteristics of the kit, the CT value is 28-30, and PCR-grade water is used as 1: 109And (5) diluting by times.
3) Subpackaging: and (4) subpackaging the calibrated positive control 50 ul/bottle into penicillin bottles.
4) Half plugging: the pipe cover is put on the pipe orifice without fastening and compacting.
5) Vacuum freeze drying: pre-freezing for 3 hours at-35 ℃ in a freeze dryer. The pressure in the freeze dryer is reduced to below 10Pa and the vacuum treatment is carried out for 2 hours at the temperature of-35 ℃. The temperature inside the freeze dryer was raised to 10 ℃ under vacuum and vacuum-treated for 2 hours. The temperature inside the freeze dryer was raised to 30 ℃ under vacuum for 2 hours.
6) And (4) capping: and clicking an automatic button to execute capping operation after the freezing process is finished.
Preparation of the lyophilized solid-state negative control RNA:
in the same way, a section of RNA of non SARS-CoV-2 is synthesized in vitro by the above method, diluted, freeze-dried and stored at-20 ℃ for standby, and the program is the same as the positive control.
Assembly of the kit
1) Putting a vacuum aluminum foil bag of SARS-CoV-2RT-PCRMix into a packaging box with water resistance and pressure resistance, putting a vial containing lyophilized positive control penicillin, putting a vial containing lyophilized negative control penicillin, and putting a vial containing reconstituted Buffer RB.
2) Putting the instruction into the use instruction.
3) And (6) packaging.
The use method of the kit comprises the following steps:
(1) preparing a reagent: adding 15 mu L of redissolving Buffer RB into each PCR tube with the freeze-dried particles, and adding 50ul of redissolving Buffer RB into freeze-dried penicillin bottles of positive control and negative control;
(2) sample and control addition: according to the number of experimental samples, 5ul of the extracted sample RNA is added into each PCR tube, 5ul of the redissolved negative and positive controls are added into the corresponding PCR tube, and the cover of the PCR tube is buckled.
(3) Centrifuging: and (3) centrifuging the PCR tube for a short time, and then putting the tube into a fluorescent quantitative PCR instrument.
(4) Setting PCR parameters: selecting FAM, HEX and Cy5 fluorescence collection channels respectively corresponding to ORF1ab, N gene and internal reference RNAse P genes; the PCR amplification program is 15min at 50 ℃ and 3min at 95 ℃, and the cycle is once; the cycle was 45 times at 95 ℃ for 15s and 60 ℃ for 60s, and fluorescence was collected at 60 ℃ for 40 minutes over the course.
(5) Determination of test results
The test is established when the negative control has no expansion curve on both FAM and HEX channels and the positive control has CT values <38 on FAM and HEX channels. When the CT value of the sample is less than or equal to 40 and the sample has a typical S-shaped amplification curve, the sample is judged to be positive; when the CT value of the sample is greater than 40 and no typical S-shaped curve exists, the sample is judged to be negative. The specific detection results were determined according to table 1.
Table 1: determination standard of SARS-CoV-2 full premixed freeze-drying fluorescence PCR detection kit
Figure BDA0002862921740000071
The properties of the reagents of the invention are further illustrated below in connection with the experiments.
S1: analytical sensitivity test (minimum test limit test)
1) Positive controls with CT value of 30.25 were serially diluted 10-fold with PCR grade water to obtain corresponding copy number concentrations of 1.0X 104、1.0×103、1.0×102、1.0×101RNA samples of copies/ul;
2) and (3) carrying out fluorescence RT-PCR detection by taking the obtained positive control NRA with 4 dilutions as a template, taking out SARS-CoV-2RT-PCRMix reagent, respectively adding 20ul of complex solution into each tube, respectively adding 5ul of corresponding RNA template after the dry powder in each tube is completely dissolved, and carrying out fluorescence RT-PCR reaction according to the instruction.
3) After the reaction is finished, the CT value is calculated.
4) As a result, the minimum detection limit was 10copies/ul, as shown in FIG. 1.
S2: assay specificity test
1) Various actual nucleic acid samples extracted by the RNA extraction kit are taken, and the actual nucleic acid samples comprise influenza A virus, influenza B virus, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, respiratory syncytial virus, parainfluenza virus, rhinovirus and the like.
2) Respectively taking out SARS-CoV-2RT-PCR Mix reagents, respectively adding 20ul of the compound solution into each tube, respectively adding corresponding RNA to dilute the sample template by 5ul when the dry powder in the tube is completely dissolved, respectively taking another 2 tubes to respectively perform negative control and positive control, respectively adding corresponding control reagents by 5ul, fully mixing uniformly, tightly covering, putting into a fluorescent quantitative PCR instrument, and setting a detection channel and an RT-PCR reaction program.
3) After the reaction is finished, the result is automatically analyzed.
The results show that the detection results corresponding to the actual nucleic acid samples are negative through the detection of the invention, and the negative and positive control results are consistent with the expected results. It can be seen that the invention has better specificity, can effectively identify SARS-CoV-2ORF1ab and N gene, and the result is shown in figure 2.
S3 Rapid detection of SARS-CoV-2 in a sample
1) Sample treatment, namely taking a sample to be detected, and extracting by using a virus RNA extraction kit to obtain a corresponding RNA detection sample;
2) adding 20ul of redissolution buffer into each PCR tube, and adding 5ul of the RNA detection sample when the dry powder in the tube is completely dissolved; another 2 tubes were used for negative and positive controls, and 5ul of corresponding control reagent was added.
3) Setting PCR reaction time and detection channels according to the instruction, and judging the result according to the judgment standard in the instruction after the reaction is finished.
The above description is only an example of the present invention, and is not intended to limit the present invention in any way, and those skilled in the art can make many variations and modifications of the present invention without departing from the scope of the present invention by using the method disclosed above, and the present invention is covered by the claims.

Claims (8)

  1. SARS-CoV-2 full premix freeze-drying fluorescence PCR detection reagent box, its characteristic is: the primer set comprises a freeze-dried solid SARS-CoV-2RT-PCR Mix, a liquid redissolving Buffer, a freeze-dried solid positive control and a freeze-dried solid negative control, wherein the freeze-dried solid SARS-CoV-2RT-PCR Mix contains a SARS-CoV-2 specific gene ORF1ab, an N gene and a primer set corresponding to a primer sequence of a human source internal reference gene RNAse P.
  2. 2. The SARS-CoV-2 full premix freeze-dried fluorescence PCR detection kit as claimed in claim 1, wherein: the primer group sequences corresponding to the SARS-CoV-2 specific ORF1ab gene and the N gene are as follows:
    ORF1ab-F：AAAGGTTATGGCTGTAGTTG
    ORF1ab-R：CGATTGTGCATCAGCTGACTG
    ORF1ab-P：TCAACTCCGCGAACCCATGCTT
    n gene-F: GTCAAGCCTCTTCTCGTTCC
    N gene-R: GGAGAAGTTCCCCTACTGC
    N gene-P: TTGAACTGTTGCGACTACGTGAT
    The primer group corresponding to the primer sequence of the human reference gene RNAse P is as follows:
    RNAse P-F：AGATTTGGACCTGCGAGCG
    RNAse P-R：GAGCGGCTGTCTCCACAAGT
    RNAse P-P：TTCTGACCTGAAGGCTCTGCGCG。
  3. 3. the SARS-CoV-2 full premix freeze-dried fluorescence PCR detection kit as claimed in claim 1, wherein: the freeze-dried solid SARS-CoV-2RT-PCR Mix contains 0.1-0.8 mmol/L dNTP, 0.01-0.1U/mu LTaq DNA polymerase, 1-10U/mu LMMLV reverse transcriptase, 0.1-2U/mu L RNase inhibitor, 0.01-0.06U/mu LUDG enzyme, 0.2-0.9 mu mol/L ORF1ab, 0.2-0.9 mu mol/LN gene, mu mol/LRNase P primer, probe, 3-6% trehalose, 0.01-0.1% glycine, DTT and 0.1-1% bovine serum albumin before freeze-drying, and the freeze-dried protective agent is prepared.
  4. 4. The SARS-CoV-2 full premix freeze-dried fluorescence PCR detection kit as claimed in claim 1, wherein: the composite solution contains 3-10 mmol/L MgSO4, 7-13 mmol/L KCl, 13-22 mmol/L LTris-HCl, 8-15 mmol/L (NH4)2SO4, 0.1-0.3% Triton X-100 and 0.3-1.6 mol/L betaine.
  5. 5. The SARS-CoV-2 full premix freeze-dried fluorescence PCR detection kit as claimed in claim 1, wherein: the positive control is lyophilized solid RNA corresponding to SARS-CoV-2 specific gene ORF1ab and N synthesized in vitro, and the negative control is purified non-SARS-CoV-2 artificially synthesized lyophilized solid RNA.
  6. The SARS-CoV-2 full premix freeze-drying fluorescence PCR detection reagent method is characterized in that: the method comprises the following steps:
    (1) preparing a reagent: adding 15 mu L of redissolving Buffer RB into each PCR tube with the freeze-dried particles, and adding 50ul of redissolving Buffer RB into freeze-dried penicillin bottles of positive control and negative control;
    (2) sample and control addition: adding 5ul of extracted sample RNA into each PCR tube according to the number of experimental samples, adding 5ul of redissolved negative and positive controls into the corresponding PCR tube, and covering the PCR tube;
    (3) centrifuging: and (3) centrifuging the PCR tube for a short time, and then putting the tube into a fluorescent quantitative PCR instrument.
    (4) Setting PCR parameters: simultaneously selecting FAM, HEX and Cy5 channels from the instrument channel to respectively correspond to ORF1ab, N gene and internal reference RNAse P genes;
    (5) and (4) carrying out PCR result analysis, verifying whether the test is established and judging the result.
  7. 7. The SARS-CoV-2 full premix freeze-dried fluorescence PCR detection reagent method of claim 6, characterized in that: after the PCR parameters in the step (4) are set, the PCR amplification procedure is carried out for 15min at 50 ℃ and 3min at 95 ℃, and the cycle is once; the fluorescence was collected at 60 ℃ after 45 cycles of 95 ℃ for 15s and 60 ℃ for 60 s.
  8. 8. The SARS-CoV-2 full premix freeze-dried fluorescence PCR detection reagent method of claim 6, characterized in that: the specific step of determining the result in the step (5) is that the test is established when the negative control has no expansion curve in both FAM and HEX channels and the CT value of the positive control in FAM and HEX channels is less than 38. When the CT value of the sample is less than or equal to 40 and the sample has a typical S-shaped amplification curve, the sample is judged to be positive; when the CT value of the sample is greater than 40 and no typical S-shaped curve exists, the sample is judged to be negative.
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CN112553380A (en) * 2020-12-31 2021-03-26 哈尔滨星云医学检验所有限公司 Method for rapidly detecting 12 respiratory viruses by utilizing multiplex PCR (polymerase chain reaction) technology and application thereof
CN113736919A (en) * 2021-09-14 2021-12-03 武汉明德生物科技股份有限公司 Nucleic acid detection kit and use method thereof

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* Cited by examiner, † Cited by third party
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CN113736919A (en) * 2021-09-14 2021-12-03 武汉明德生物科技股份有限公司 Nucleic acid detection kit and use method thereof

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