CN114317822A - Multiplex fluorescence quantitative PCR method for detecting new coronavirus nucleic acid - Google Patents
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- 238000000034 method Methods 0.000 title claims abstract description 61
- 241000711573 Coronaviridae Species 0.000 title claims abstract description 39
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 22
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 22
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 22
- 238000003753 real-time PCR Methods 0.000 title claims abstract description 17
- 239000000523 sample Substances 0.000 claims abstract description 85
- 230000003321 amplification Effects 0.000 claims abstract description 39
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 39
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 37
- 238000001514 detection method Methods 0.000 claims abstract description 32
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- 230000003612 virological effect Effects 0.000 claims abstract description 9
- 239000002773 nucleotide Substances 0.000 claims description 18
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- 238000002474 experimental method Methods 0.000 claims description 10
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 3
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- 238000011529 RT qPCR Methods 0.000 claims 4
- 230000008685 targeting Effects 0.000 abstract description 6
- 239000013642 negative control Substances 0.000 abstract description 4
- 239000013641 positive control Substances 0.000 abstract description 4
- 108700002099 Coronavirus Nucleocapsid Proteins Proteins 0.000 abstract description 2
- 230000035945 sensitivity Effects 0.000 description 8
- 108020004414 DNA Proteins 0.000 description 6
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- 230000000694 effects Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 208000031504 Asymptomatic Infections Diseases 0.000 description 1
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- 230000002265 prevention Effects 0.000 description 1
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a multiple fluorescence quantitative PCR method for detecting new coronavirus nucleic acid. The technical system of the invention consists of two pairs of primer probes respectively aiming at new coronavirus ORF1ab and N genes, wherein each pair of primer probes consists of an upstream primer, a downstream primer and a TaqMan probe. Wherein the primer probe targeting the new coronavirus ORF1ab gene is matched with the nt-nt region of the viral genome, and the primer probe targeting the new coronavirus N gene is matched with the nt-nt region of the viral genome. The two sets of primer probes form a multiplex fluorescence quantitative PCR method, and a positive control and a negative control are arranged. And (3) taking the fluorescent quantitative PCR amplification curve as a detection result judgment basis, and when the positive control amplification curve is positive and the negative control has no typical amplification curve, the experimental result of the same-plate detection is credible.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a multiple fluorescence quantitative PCR method for detecting new coronavirus nucleic acid.
Background
The qRT-PCR (fluorescent quantitative reverse transcription PCR) method is a common method for detecting the nucleic acid of the novel coronavirus, and has the advantages of high sensitivity, good specificity, large detection flux, convenience for large-scale equipment and the like [1 ]. The multiplex qRT-PCR method utilizes multiple fluorescence labeled different TaqMan probes, and can simultaneously detect multiple targets in the same reaction tube [2 ]. The effect of the qRT-PCR method for detecting the new coronavirus nucleic acid is influenced by various factors, including the performances of primers and TaqMan probes, the quality of reagents and consumables, detection equipment, operators, environmental conditions and the like. Wherein the performance of primers and probes is a major influencing factor for the detection of viral nucleic acids [3 ]. Due to the complex secondary structure and GC content difference of the viral genome, only a limited region is available for designing a detection primer probe. The core of establishing a virus nucleic acid detection qRT-PCR method is to select which section of the virus genome designs a primer probe and perform methodology optimization according to the primer probe. The reason for the performance difference of qRT-PCR products for detecting new coronavirus nucleic acid developed by different units is mainly derived from the advantages and disadvantages of primer probe sequences.
Asymptomatic infection has become an important feature of new coronavirus epidemic spread, and is characterized by no obvious clinical symptoms after infection, low virus load and the need of continuous and multiple detections to discover virus infection [4 ]. The performance of the new coronavirus nucleic acid detection method directly influences the epidemic situation prevention and control effect, so that the research and development of a new coronavirus nucleic acid detection reagent with higher sensitivity and better performance are urgently needed.
Reference documents:
[1]Wang HL,Li G,Zhao J,Li YJ,Ai YS.An Overview ofNucleic Acid Testing forthe Novel Coronavirus SARS-CoV-2.FrontMed(Lausanne),2020,7:571709.
[2]Perchetti GA,Nalla AK,Huang ML,Jerome KR,Greninger AL. Multiplexing primer/probe sets for detection of SARS-CoV-2by qRT-PCR.J Clin Virol,2020,129:1044499.
[3]Zhou Y,Zhang L,Xie YH,Wu J.Advancements in detection of SARS-CoV-2infection for confronting COVID-19pandemics.Lab Invest,2021,DOI: 10.1038/s41374-021-00663-w.
[4]Oran DP,Topol EJ.The Proportion of SARS-CoV-2Infections That Are Asymptomatic.Ann Intern Med,2021,174(5):655-662.
disclosure of Invention
Aiming at the problems, the invention provides a multiplex fluorescence quantitative PCR method for detecting new coronavirus nucleic acid.
In order to achieve the purpose, the invention adopts the following technical scheme:
a TaqMan probe method and a qRT-PCR method are adopted, detection primers and probe sequences are designed and optimized based on a new coronavirus genome conserved region, the optimal combination is a multiple qRT-PCR method, and high-sensitivity, high-throughput and low-cost detection of new coronavirus nucleic acid is realized by extracting virus nucleic acid and performing qRT-PCR on a 96-hole or 384-hole reaction plate by means of the characteristic of high sensitivity of the TaqMan probe.
The technical system of the method consists of two pairs of primer probes respectively aiming at the new coronavirus ORF1ab and N genes, wherein each pair of primer probes consists of an upstream primer, a downstream primer and a TaqMan probe. Wherein the primer probe targeting the new coronavirus ORF1ab gene is matched with the region of the viral genome nt 13756-nt 13976 (taking the genome sequence NC-045512.2 of the new coronavirus prototype strain as reference, the same below), and the primer probe targeting the new coronavirus N gene is matched with the region of the viral genome nt 28757-nt 28907. The probe targeting ORF1ab gene labels 5 '-FAM and BHQ 1-3' fluorophores, and the probe targeting N gene labels 5 '-VIC and BHQ 1-3' fluorophores. The two sets of primer probes form a multiplex fluorescence quantitative PCR method, and a positive control and a negative control are arranged. And (3) taking the fluorescent quantitative PCR amplification curve as a detection result judgment basis, and when the positive control amplification curve is positive and the negative control has no typical amplification curve, the experimental result of the same-plate detection is credible.
A multiplex fluorescent quantitative PCR method for detecting new coronavirus nucleic acid comprises the following steps:
step 1, extracting new coronavirus RNA by using a Trizol method;
step 2, designing a qRT-PCR primer probe based on a TaqMan probe:
step 2.1, primer design: respectively designing an upstream primer pair and a downstream primer pair by combining nucleotide sequences of novel coronavirus ORF1ab and N gene conserved regions according to a general principle of primer design;
when the ORF1ab gene target is detected, the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 1: 5'-ATAGACGGTGACATGGTACCAC-3', the nucleotide sequence of the downstream primer is shown in SEQ ID NO. 2: 5'-CCTAAGTTGGCGTATACGCG-3', respectively;
when detecting the N gene target, the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 3: 5'-CCTCAAGGAACAACATTGCC-3', the nucleotide sequence of the downstream primer is shown in SEQ ID NO. 4: 5'-CAGCCATTCTAGCAGGAGAAG-3', respectively;
step 2.2, designing a TaqMan probe: designing a TaqMan probe according to a general principle of TaqMan probe design and combining the sequence characteristics of the viral genome region limited by the upstream and downstream primers in the step 2.1;
when ORF1ab gene target is detected, the nucleotide sequence of the TaqMan probe is shown in SEQ ID NO. 5: 5'-TACACAATGGCAGACCTCGTCTATGCT-3', respectively;
when detecting the N gene target, the nucleotide sequence of the TaqMan probe is shown as SEQ ID NO. 6: 5'-CAAGCCTCTTCTCGTTCCTCATCACGT-3', respectively;
step 3, performing qRT-PCR experiment based on a TaqMan probe;
and 4, interpretation of results:
step 4.1, judging the sample to be positive when the Ct is less than 35 and the sample has a typical amplification curve;
and 4.2, judging the sample to be suspicious positive when the Ct is less than 35 but does not have a typical amplification curve, and testing again by using the same method: if the amplification curve is still atypical, judging the amplification curve to be negative; if the Ct is less than 35 and the amplification curve is typical, the amplification curve is judged to be positive;
and 4.3, judging the sample to be suspicious positive when the Ct is greater than 35 and the sample has a typical amplification curve, and testing again by using the same method: if the result is still Ct >35 and has a typical amplification curve, the result is judged to be positive; if no typical amplification curve exists, judging the amplification curve to be negative;
step 4.4, when the Ct is more than 35 but does not have a typical amplification curve, judging that the sample is negative;
step 4.5, according to the result judgment method of 4.1-4.4, when the two targets of ORF1ab and N are positive, judging that the clinical sample is positive to the new coronavirus;
step 4.6, according to the result judgment method of 4.1-4.4, when the two targets of ORF1ab and N are negative, judging that the clinical sample is negative to the new coronavirus;
and 4.7, according to the result judgment method of 4.1-4.4, when one of the two targets of ORF1ab and N is positive and the other target is negative, the same method is applied to test again, and if the same result is still obtained, other new coronavirus nucleic acid detection methods are required to be used for rechecking.
Further, the step 3 of qRT-PCR experiment based on TaqMan probe method comprises the following specific steps:
step 3.1, preparing a reaction system: each reaction system has the volume of 20 mu L, and contains 10 mu L of 2X reaction liquid, 0.2 mu L of 50 mu M upstream primer, 0.2 mu L of 50 mu M downstream primer, 0.1 mu L of 50 mu M TaqMan probe, 7 mu L of sterile RNase-free water and 2 mu L of RNA template, and each reaction system contains two sets of primer pairs and probes aiming at ORF1ab and N genes;
step 3.2, qRT-PCR reaction conditions: the following reaction procedure was performed on a fluorescent quantitative PCR instrument: at 95 ℃ for 3 minutes; 45 cycles of 95 deg.C, 30 seconds to 60 deg.C, 60 seconds.
Compared with the prior art, the invention has the following advantages:
1. and the detection sensitivity is improved. The new coronavirus nucleic acid is detected based on a TaqMan probe method qRT-PCR, and the detection sensitivity can reach 1 copy/microliter.
2. The detection cost is reduced, and the detection flux is improved. The technology combines the primer probe aiming at the two targets of the new coronavirus ORF1ab and the N gene into a set of multiplex fluorescence quantitative PCR method, can simultaneously detect the two gene targets of the new coronavirus in one reaction tube, saves the consumption of reagent consumables while ensuring the reliability of a detection result, improves the use efficiency of a detection instrument, can detect more samples under the same condition, and thus improves the detection flux.
Drawings
FIG. 1 is a graph showing the results of the sensitivity test in the method of the present invention.
Detailed Description
Example 1
A multiplex fluorescent quantitative PCR method for detecting new coronavirus nucleic acid comprises the following steps:
step 1, extracting new coronavirus RNA by using a Trizol method;
step 2, designing a qRT-PCR primer probe based on a TaqMan probe:
step 2.1, primer design: respectively designing an upstream primer pair and a downstream primer pair by combining nucleotide sequences of novel coronavirus ORF1ab and N gene conserved regions according to a general principle of primer design;
when the ORF1ab gene target is detected, the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 1: 5'-ATAGACGGTGACATGGTACCAC-3', the nucleotide sequence of the downstream primer is shown in SEQ ID NO. 2: 5'-CCTAAGTTGGCGTATACGCG-3', respectively;
when detecting the N gene target, the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 3: 5'-CCTCAAGGAACAACATTGCC-3', the nucleotide sequence of the downstream primer is shown in SEQ ID NO. 4: 5'-CAGCCATTCTAGCAGGAGAAG-3', respectively;
step 2.2, designing a TaqMan probe: designing a TaqMan probe according to a general principle of TaqMan probe design and combining the sequence characteristics of the viral genome region limited by the upstream and downstream primers in the step 2.1;
when ORF1ab gene target is detected, the nucleotide sequence of the TaqMan probe is shown in SEQ ID NO. 5: 5'-TACACAATGGCAGACCTCGTCTATGCT-3', respectively;
when detecting the N gene target, the nucleotide sequence of the TaqMan probe is shown as SEQ ID NO. 6: 5'-CAAGCCTCTTCTCGTTCCTCATCACGT-3', respectively;
and 3, performing qRT-PCR experiment based on a TaqMan probe, and specifically comprising the following steps:
step 3.1, preparing a reaction system: each reaction system has the volume of 20 mu L, and contains 10 mu L of 2X reaction liquid, 0.2 mu L of 50 mu M upstream primer, 0.2 mu L of 50 mu M downstream primer, 0.1 mu L of 50 mu M TaqMan probe, 7 mu L of sterile RNase-free water and 2 mu L of RNA template, and each reaction system contains two sets of primer pairs and probes aiming at ORF1ab and N genes;
step 3.2, qRT-PCR reaction conditions: the following reaction procedure was performed on a fluorescent quantitative PCR instrument: at 95 ℃ for 3 minutes; 45 cycles of 95 deg.C, 30 seconds-60 deg.C, 60 seconds
And 4, interpretation of results:
step 4.1, judging the sample to be positive when the Ct is less than 35 and the sample has a typical amplification curve;
and 4.2, judging the sample to be suspicious positive when the Ct is less than 35 but does not have a typical amplification curve, and testing again by using the same method: if the amplification curve is still atypical, judging the amplification curve to be negative; if the Ct is less than 35 and the amplification curve is typical, the amplification curve is judged to be positive;
and 4.3, judging the sample to be suspicious positive when the Ct is greater than 35 and the sample has a typical amplification curve, and testing again by using the same method: if the result is still Ct >35 and has a typical amplification curve, the result is judged to be positive; if no typical amplification curve exists, judging the amplification curve to be negative;
step 4.4, when the Ct is more than 35 but does not have a typical amplification curve, judging that the sample is negative;
step 4.5, according to the result judgment method of 4.1-4.4, when the two targets of ORF1ab and N are positive, judging that the clinical sample is positive to the new coronavirus;
step 4.6, according to the result judgment method of 4.1-4.4, when the two targets of ORF1ab and N are negative, judging that the clinical sample is negative to the new coronavirus;
and 4.7, according to the result judgment method of 4.1-4.4, when one of the two targets of ORF1ab and N is positive and the other target is negative, the same method is applied to test again, and if the same result is still obtained, other new coronavirus nucleic acid detection methods are required to be used for rechecking.
Example 2
Sensitivity experiments were performed on the method of the invention (template dilution 10)8-10-1Copy/microliter), the results of the experiment are shown in FIG. 1, and when ORF1ab gene was detected, detectiveSensitivity of 1.25 copies/microliter; when the N gene was detected, the detection sensitivity was 0.89 copy/microliter.
Example 3
The precision of the evaluation method of the repeatability experiment refers to the closeness degree between a series of single measurement values obtained by repeatedly measuring the same sample for many times under a certain condition, is an index of the random error magnitude of the reaction, and is divided into an intra-batch repeatability experiment and an inter-batch repeatability experiment.
We examined the reproducibility (precision) of the method, measured by the coefficient of variation, and the results are shown in Table 1(ORF1ab gene) and Table 2(N gene).
TABLE 1 repeatability (precision) of the process
TABLE 2 repeatability (precision) of the process
The calculation method of the variation coefficient comprises the following steps:
coefficient of variation (%) - (Ct standard deviation/Ct average) × 100%
As can be seen from tables 1 and 2: the intra-batch experimental variation coefficient range of ORF1ab gene is 0.22-1.18%, and the inter-batch experimental variation coefficient range is 0.45-1.47%;
the experimental variation coefficient range in the N gene batch is 0.53-1.89%, and the experimental variation coefficient range between batches is 1.66-1.92%;
the experimental variation coefficients of the gene detection primer probe in batch and between batches are less than 2 percent. Therefore, the method has good repeatability (precision).
Example 4
The linear and range analysis method has the advantages that the linearity is the capability of acquiring the experimental result which is in direct proportion to the concentration of the test substance in the sample in a given range, namely, the concentration range of the test substance which is in linear relation with the experimental result, and is an important index of the detection performance of the reaction method.
We examined the linear detection range of the method, and the experimental results are shown in Table 3(ORF1ab gene) and Table 4(N gene). Wherein the slope is known, the system of the method is in the template 106-101The copy/microliter can obtain a detection signal which is in linear relation with the template amount, which shows that the method has a wide linear detection range.
TABLE 3 Linear detection Range of ORF1ab Gene in this method
TABLE 4 Linear detection Range of the method for the N gene
Sequence listing
SEQ ID NO.1：
5’-ATAGACGGTGACATGGTACCAC-3’
SEQ ID NO.2：
5’-CCTAAGTTGGCGTATACGCG-3’
SEQ ID NO.3：
5’-CCTCAAGGAACAACATTGCC-3’
SEQ ID NO.4：
5’-CAGCCATTCTAGCAGGAGAAG-3’
SEQ ID NO.5：
5’-TACACAATGGCAGACCTCGTCTATGCT-3’
SEQ ID NO.6：
5’-CAAGCCTCTTCTCGTTCCTCATCACGT-3’
Sequence listing
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Claims (2)
1. A multiplex fluorescent quantitative PCR method for detecting new coronavirus nucleic acid is characterized by comprising the following steps:
step 1, extracting new coronavirus RNA by using a Trizol method;
step 2, designing a qRT-PCR primer probe based on a TaqMan probe:
step 2.1, primer design: respectively designing an upstream primer pair and a downstream primer pair by combining nucleotide sequences of novel coronavirus ORF1ab and N gene conserved regions according to a general principle of primer design;
when the ORF1ab gene target is detected, the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 1: 5'-ATAGACGGTGACATGGTACCAC-3', the nucleotide sequence of the downstream primer is shown in SEQ ID NO. 2: 5'-CCTAAGTTGGCGTATACGCG-3', respectively;
when detecting the N gene target, the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 3: 5'-CCTCAAGGAACAACATTGCC-3', the nucleotide sequence of the downstream primer is shown in SEQ ID NO. 4: 5'-CAGCCATTCTAGCAGGAGAAG-3', respectively;
step 2.2, designing a TaqMan probe: designing a TaqMan probe according to a general principle of TaqMan probe design and combining the sequence characteristics of the viral genome region limited by the upstream and downstream primers in the step 2.1;
when ORF1ab gene target is detected, the nucleotide sequence of the TaqMan probe is shown in SEQ ID NO. 5: 5'-TACACAATGGCAGACCTCGTCTATGCT-3', respectively;
when detecting the N gene target, the nucleotide sequence of the TaqMan probe is shown as SEQ ID NO. 6: 5'-CAAGCCTCTTCTCGTTCCTCATCACGT-3', respectively;
step 3, performing qRT-PCR experiment based on a TaqMan probe;
and 4, interpretation of results:
step 4.1, judging the sample to be positive when the Ct is less than 35 and the sample has a typical amplification curve;
and 4.2, judging the sample to be suspicious positive when the Ct is less than 35 but does not have a typical amplification curve, and testing again by using the same method: if the amplification curve is still atypical, judging the amplification curve to be negative; if the Ct is less than 35 and the amplification curve is typical, the amplification curve is judged to be positive;
and 4.3, judging the sample to be suspicious positive when the Ct is greater than 35 and the sample has a typical amplification curve, and testing again by using the same method: if the result is still Ct >35 and has a typical amplification curve, the result is judged to be positive; if no typical amplification curve exists, judging the amplification curve to be negative;
step 4.4, when the Ct is more than 35 but does not have a typical amplification curve, judging that the sample is negative;
step 4.5, according to the result judgment method of 4.1-4.4, when the two targets of ORF1ab and N are positive, judging that the clinical sample is positive to the new coronavirus;
step 4.6, according to the result judgment method of 4.1-4.4, when the two targets of ORF1ab and N are negative, judging that the clinical sample is negative to the new coronavirus;
and 4.7, according to the result judgment method of 4.1-4.4, when one of the two targets of ORF1ab and N is positive and the other target is negative, the same method is applied to test again, and if the same result is still obtained, other new coronavirus nucleic acid detection methods are required to be used for rechecking.
2. The multiplex fluorescent quantitative PCR method for detecting the neocoronavirus nucleic acid according to claim 1, wherein the qRT-PCR experiment based on the TaqMan probe method in the step 3 comprises the following specific steps:
step 3.1, preparing a reaction system: each reaction system has the volume of 20 mu L, and contains 10 mu L of 2X reaction liquid, 0.2 mu L of 50 mu M upstream primer, 0.2 mu L of 50 mu M downstream primer, 0.1 mu L of 50 mu M TaqMan probe, 7 mu L of sterile RNase-free water and 2 mu L of RNA template, and each reaction system contains two sets of primer pairs and probes aiming at ORF1ab and N genes;
step 3.2, qRT-PCR reaction conditions: the following reaction procedure was performed on a fluorescent quantitative PCR instrument: at 95 ℃ for 3 minutes; 45 cycles of 95 deg.C, 30 seconds to 60 deg.C, 60 seconds.
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| CN111378787A (en) * | 2020-04-08 | 2020-07-07 | 山西医科大学 | Novel coronavirus detection method |
| CN111733295A (en) * | 2020-07-31 | 2020-10-02 | 广州领上源生物科技有限公司 | Primer group and kit for detecting novel coronavirus |
| CN112662808A (en) * | 2020-12-02 | 2021-04-16 | 湖州市中心医院 | Novel coronavirus COVID-19 nucleic acid detection kit and detection method thereof |
| CN112725537A (en) * | 2020-03-12 | 2021-04-30 | 宁波海尔施基因科技有限公司 | Multiplex real-time fluorescent quantitative PCR (polymerase chain reaction) kit and method for detecting 2019 novel coronavirus and primer probe composition |
| CN113322348A (en) * | 2021-03-27 | 2021-08-31 | 中国人民解放军军事科学院军事医学研究院 | High-sensitivity novel coronavirus 2019-nCoV nucleic acid detection kit and use method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112725537A (en) * | 2020-03-12 | 2021-04-30 | 宁波海尔施基因科技有限公司 | Multiplex real-time fluorescent quantitative PCR (polymerase chain reaction) kit and method for detecting 2019 novel coronavirus and primer probe composition |
| CN111378787A (en) * | 2020-04-08 | 2020-07-07 | 山西医科大学 | Novel coronavirus detection method |
| CN111733295A (en) * | 2020-07-31 | 2020-10-02 | 广州领上源生物科技有限公司 | Primer group and kit for detecting novel coronavirus |
| CN112662808A (en) * | 2020-12-02 | 2021-04-16 | 湖州市中心医院 | Novel coronavirus COVID-19 nucleic acid detection kit and detection method thereof |
| CN113322348A (en) * | 2021-03-27 | 2021-08-31 | 中国人民解放军军事科学院军事医学研究院 | High-sensitivity novel coronavirus 2019-nCoV nucleic acid detection kit and use method thereof |
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