CN114164303A - qRT-PCR method for identifying novel coronavirus Gamma variant strain - Google Patents
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a qRT-PCR method for identifying a novel coronavirus Gamma variant strain. In the method, a TaqMan probe is introduced for improving the detection sensitivity and specificity; in order to reduce the cost, 2 pairs of primers are changed into 1 pair of half primers, namely: 2 upstream primers respectively target mutation sites and original non-mutation sites, and 1 downstream primer is shared by 2 upstream primers. In order to enhance the sensitivity of mutation detection, mutation is introduced at the 3 rd site in the 5' direction of the upstream mutation primer mutation site, and the degree of matching between the mutation primer and the non-variant viral nucleic acid and between the non-variant primer and the variant viral nucleic acid in the reaction system is reduced, so that the amplification curve of the variant primer amplified variant viral nucleic acid in the reaction system is earlier than that of the non-variant nucleic acid amplified, and similarly, the amplification curve of the non-variant primer amplified non-variant nucleic acid is earlier than that of the amplified variant nucleic acid.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a qRT-PCR method for identifying a novel coronavirus Gamma variant strain.
Background
Gene sequencing is the most common technique for identifying variants of coronavirus and has the following disadvantages [1 ]]: 1. the time from sample to result is long, and the uncontrollable influence factors are many. The time from sample processing to result reporting is 6-8 hours, and most virus detection units do not have gene sequencing conditions and need to be handed to a third-party sequencing company for generation and testing, and the sample road transportation and possible computer queuing waiting time lead the gene sequencing not only to be slow, but also to have a plurality of uncontrollable influence factors; 2. the detection sensitivity is low. Before the gene sequencing is tested on a computer, gene amplification is needed, and sequencing can be influenced if the amplification effect of a low-abundance nucleic acid sample is slightly poor; 3. the detection cost is high. The sequencing of the new coronavirus gene comprises complicated steps of sample nucleic acid extraction, RT-PCR, PCR product purification, on-machine testing and the like, so that the detection material and labor cost are high, and the deviation of the detection result is easily caused by careless operation in multi-step operation. The defects enable gene sequencing to influence the detection timeliness, flux and cost of virus variant strains when being applied to the prevention and control of the new coronary pneumonia epidemic situation, possibly influence the detection result and have important influence on the prevention and control work of the new coronary pneumonia epidemic situation[2]。
The new coronavirus Gamma variant (also called p.1 branch) was first discovered in brazil 1 month in 2021, and varied at the following sites of the virus Spike protein: K417T, E484K and N501Y [3 ]]Has higher transmission power and neutralizing antibody antagonism than the original strain, alpha variant strain and Beta variant strain virus, and is possible to antagonize the protective action of the vaccine[3]. The work of preventing and controlling the new crown epidemic situation needs a rapid detection method of the new crown virus Gamma variant strain.
Reference documents:
[1]Bezerra,et al.A Sanger-based approach for scaling up screening of SARS-CoV-2 variants of interest and concern.Infection,Genetics and Evolution,2021,92:104910.
[2]Vega-Magana,et al.RT-qPCR Assays for Rapid Detection of the N501Y,69-70del,K417N,and E484K SARS-CoV-2 Mutations:A Screening Strategy to Identify Variants with Clinical Impact.Frontiers in Cellular and Infection Microbiology,2021,11:672562.
[3]Nonaka,et al.SARS-CoV-2 variant of concern P.1(Gamma)infection in young and middle-aged patients admitted to the intensive care units of a single hospital in Salvador,Northeast Brazil,February 2021.International journal of infectious diseases,2021,111:47-54.
disclosure of Invention
Aiming at the problems, the invention provides a qRT-PCR method for identifying a novel coronavirus Gamma variant strain.
In order to achieve the purpose, the invention adopts the following technical scheme:
by adopting an improved PCR method named an Amplification Retardation Mutation System (ARMS), the virus variant can be accurately identified by using fluorescent quantitative PCR instruments equipped by most detection units through complicated operation steps of virus nucleic acid extraction-qRT-PCR to replace gene sequencing, virus nucleic acid extraction-RT-PCR amplification-PCR product purification-on-machine test and the like, the dependence on expensive sequencers is overcome, and the universality of the detection method of the new coronavirus variant is improved. The high-sensitivity, high-flux and low-cost detection of the new coronavirus variant is realized by a 96-hole or 384-hole qRT-PCR reaction plate and the high-sensitivity and low-cost point of the qRT-PCR by a TaqMan probe method.
The basic method technology system consists of 2 pairs of primers, wherein one pair of primers is combined with a mutation site, the other pair of primers is combined with an original non-mutation site, and whether the mutation exists is judged according to the existence of amplified fragments and the length of the fragments.
In the method, a TaqMan probe is introduced for improving the detection sensitivity and specificity; in order to reduce the cost, 2 pairs of primers are changed into 1 pair of half primers, namely: 2 upstream primers respectively target mutation sites and original non-mutation sites, and 1 downstream primer is shared by 2 upstream primers. In order to enhance the sensitivity of mutation detection, mutation is introduced at the 3 rd site in the 5' direction of the upstream mutation primer mutation site, and the degree of matching between the mutation primer and the non-variant viral nucleic acid and between the non-variant primer and the variant viral nucleic acid in the reaction system is reduced, so that the amplification curve of the variant primer amplified variant viral nucleic acid in the reaction system is earlier than that of the non-variant nucleic acid amplified, and similarly, the amplification curve of the non-variant primer amplified non-variant nucleic acid is earlier than that of the amplified variant nucleic acid. And (3) detecting the sample by using two sets of experimental systems of the variation primer and the non-variation primer, wherein the variation primer system is used for judging that the detection sample contains the variation virus when an amplification curve of the variation primer system is earlier than that of the non-variation primer.
A qRT-PCR method for identifying novel coronavirus Gamma variant strains 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 ARMS:
step 2.1, primer design: according to the general principle of primer design, combining nucleotide sequences near the variation sites of the novel coronavirus Indian variety, and designing two pairs of primers for each variation site, wherein the 3' end of an upstream primer is respectively matched with a variation point and a non-variation point, namely a variation upstream primer and a non-variation upstream primer, and the two pairs of upstream primers share a downstream primer;
when the mutation site to be identified is K417T, the nucleotide sequence of the mutation upstream primer is shown in SEQ ID NO. 1: 5'-CCAGGGCAAACTGGGAC-3', the nucleotide sequence of the non-variant upstream primer is shown in SEQ ID NO. 2: 5'-CCAGGGCAAACTGGAAAG-3', the downstream primer is shown in SEQ ID NO. 3: 5'-CCACCAACCTTAGAATCAAGATTG-3', respectively;
when the mutation site to be identified is E484K, the nucleotide sequence of the mutation upstream primer is shown in SEQ ID NO. 4: 5'-GCACACCTTGTAATGGTGCTA-3', the nucleotide sequence of the non-variant upstream primer is shown in SEQ ID NO. 5: 5'-GCACACCTTGTAATGGTGTTG-3', the downstream primer is shown in SEQ ID NO. 6: 5'-AGTTGCTGGTGCATGTAGAAG-3', respectively;
when the mutation site to be identified is N501Y, the nucleotide sequence of the mutation upstream primer is shown in SEQ ID NO. 7: 5'-CAATCATATGGTTTCCAACCCAGTT-3', the nucleotide sequence of the non-variant upstream primer is shown in SEQ ID NO. 8: 5'-CAATCATATGGTTTCCAACCCACTA-3', the downstream primer is shown in SEQ ID NO. 9: 5'-CAGTAAGAACACCTGTGCCTGTT-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, wherein two pairs of primers of each mutation site share one TaqMan probe;
when the mutation site to be identified is K417T, the nucleotide sequence of the TaqMan probe is shown in SEQ ID NO. 10: 5'-CCAGATGATTTTACAGGCTGCGTTATAGCTTGG-3', respectively;
when the mutation site to be identified is E484K, the nucleotide sequence of the TaqMan probe is shown in SEQ ID NO. 11: 5'-CCAACCCACTAATGGTGTTGGTTACCAACC-3', respectively;
when the mutation site to be identified is N501Y, the nucleotide sequence of the TaqMan probe is shown in SEQ ID NO. 12: 5'-CTACATGCACCAGCAACTGTTTGTGGACC-3', respectively;
step 3, performing qRT-PCR experiment based on ARMS;
and 4, interpretation of results:
step 4.1, judging that the mutation occurs in the detection sample when the occurrence time of the amplification curve of the mutation primer system is more than 2 cycles earlier than that of the non-mutation primer system;
and 4.2, judging that no variation occurs in the detection sample when the occurrence time of the amplification curve of the non-variation primer system is more than 2 cycles earlier than that of the variation primer system.
Further, the step 3 of performing qRT-PCR experiments based on ARMS comprises the following specific steps:
step 3.1, preparing a reaction system: each reaction system has the volume of 20 mu L, wherein the reaction system 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, 7.5 mu L of sterile RNase-free water and 2 mu L of RNA template, the detection of each mutation site consists of two reaction systems, one contains a mutation upstream primer, the other contains a non-mutation downstream primer, and the other components are the same;
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, 15 seconds to 60 deg.C, 30 seconds.
Compared with the prior art, the invention has the following advantages:
1. the detection speed is improved, and the dependence on expensive instruments is reduced. By applying the technology of the patent application, the identification speed of the novel coronavirus variant is shortened from 4-6 hours to 2 hours, and the technical method does not depend on an expensive sequencer and can be completed only by a common fluorescent quantitative PCR instrument, so that the dependence on expensive instruments such as the sequencer is reduced, a virus detection user can complete the identification of the coronavirus variant inside a unit, the time for sample transportation and the like is saved, and uncontrollable factors are reduced to the maximum extent;
2. and the detection sensitivity is improved. The method is characterized in that the virus nucleic acid variation is detected based on a TaqMan probe method qRT-PCR, the detection sensitivity can reach 1-10 copies/microliter, the detection of the virus nucleic acid variation can be realized by the qRT-PCR having an amplification signal, and the influence of a weak amplification signal on the detection of the nucleic acid variation based on gene sequencing is avoided.
3. The detection cost is reduced, and the detection flux is improved. The whole experimental process of the technology only comprises two steps of virus nucleic acid extraction and qRT-PCR, the experimental steps are few, the required material and labor cost is greatly reduced, and the high flux of the sample can be conveniently realized through a 96-hole or 384-hole reaction plate.
4. The operation pollution probability is small, and the high-throughput operation is easy. The existing first-generation sequencing technology needs multiple steps of virus RNA extraction, RT-PCR amplification, PCR product purification, on-machine sequencing and the like for detecting virus variation, the batch operation is easy to introduce operation pollution, the method only has two steps of virus RNA extraction and qRT-PCR operation, the corresponding operation pollution reduction probability of the steps is also reduced, and fewer operation steps are easy to realize the detection of a large number of samples at one time.
5. The performance of the primer probe of each site to be identified when used alone and in combination with other primer probes is different: when used alone, the primer is not influenced by other primer probes; when the fluorescent quantitative PCR system is combined with other primer probes to form a multiple fluorescent quantitative PCR system, the fluorescent quantitative PCR system is easily influenced by the other primer probes, and the optimal combination needs to be carried out on the aspects of the dosage of the primer probes of each site to be identified, reaction conditions, positive result judgment threshold values and the like.
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 qRT-PCR method for identifying novel coronavirus Gamma variant strains 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 ARMS:
step 2.1, primer design: according to the general principle of primer design, combining nucleotide sequences near the variation sites of the novel coronavirus Indian variety, and designing two pairs of primers for each variation site, wherein the 3' end of an upstream primer is respectively matched with a variation point and a non-variation point, namely a variation upstream primer and a non-variation upstream primer, and the two pairs of upstream primers share a downstream primer;
when the mutation site to be identified is K417T, the nucleotide sequence of the mutation upstream primer is shown in SEQ ID NO. 1: 5'-CCAGGGCAAACTGGGAC-3', the nucleotide sequence of the non-variant upstream primer is shown in SEQ ID NO. 2: 5'-CCAGGGCAAACTGGAAAG-3', the downstream primer is shown in SEQ ID NO. 3: 5'-CCACCAACCTTAGAATCAAGATTG-3', respectively;
when the mutation site to be identified is E484K, the nucleotide sequence of the mutation upstream primer is shown in SEQ ID NO. 4: 5'-GCACACCTTGTAATGGTGCTA-3', the nucleotide sequence of the non-variant upstream primer is shown in SEQ ID NO. 5: 5'-GCACACCTTGTAATGGTGTTG-3', the downstream primer is shown in SEQ ID NO. 6: 5'-AGTTGCTGGTGCATGTAGAAG-3', respectively;
when the mutation site to be identified is N501Y, the nucleotide sequence of the mutation upstream primer is shown in SEQ ID NO. 7: 5'-CAATCATATGGTTTCCAACCCAGTT-3', the nucleotide sequence of the non-variant upstream primer is shown in SEQ ID NO. 8: 5'-CAATCATATGGTTTCCAACCCACTA-3', the downstream primer is shown in SEQ ID NO. 9: 5'-CAGTAAGAACACCTGTGCCTGTT-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, wherein two pairs of primers of each mutation site share one TaqMan probe;
when the mutation site to be identified is K417T, the nucleotide sequence of the TaqMan probe is shown in SEQ ID NO. 10: 5'-CCAGATGATTTTACAGGCTGCGTTATAGCTTGG-3', respectively;
when the mutation site to be identified is E484K, the nucleotide sequence of the TaqMan probe is shown in SEQ ID NO. 11: 5'-CCAACCCACTAATGGTGTTGGTTACCAACC-3', respectively;
when the mutation site to be identified is N501Y, the nucleotide sequence of the TaqMan probe is shown in SEQ ID NO. 12: 5'-CTACATGCACCAGCAACTGTTTGTGGACC-3', respectively;
step 3, performing qRT-PCR experiment based on ARMS:
step 3.1, preparing a reaction system: each reaction system has the volume of 20 mul, wherein the reaction system contains 10 mul of 2X reaction liquid, 0.2 mul of 50 mul upstream primer, 0.2 mul of 50 mul downstream primer, 0.1 mul of 50 mul TaqMan probe, 7.5 mul of sterile RNase-free water and 2 mul RNA template, the detection of each variation site consists of two reaction systems, one contains a variation upstream primer, the other contains a non-variation downstream primer, and other components are the same;
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, 15 seconds to 60 deg.C, 30 seconds.
And 4, interpretation of results:
step 4.1, judging that the mutation occurs in the detection sample when the occurrence time of the amplification curve of the mutation primer system is more than 2 cycles earlier than that of the non-mutation primer system;
and 4.2, judging that no variation occurs in the detection sample when the occurrence time of the amplification curve of the non-variation primer system is more than 2 cycles earlier than that of the variation primer system.
Example 2
Sensitivity experiments were performed on the method of the invention(template dilution 107-100Copy/microliter), the experimental result is shown in fig. 1, when the mutation site to be identified is K417T, the detection sensitivity is 2.36 copies/microliter; when the mutation site needing to be identified is E484K, the detection sensitivity is 5.50 copies/microliter; when the mutation site to be identified is N501Y, the detection sensitivity is 4.35 copies/microliter.
Example 3
The precision of the repeatability test evaluation method refers to the closeness degree between a series of single measured values obtained by repeatedly measuring the same sample for many times under certain conditions, is an index of random error magnitude of reaction, and is divided into batch repeatability tests, batch-to-batch repeatability tests (day-to-day and day-to-day repeatability tests), operator/instrument repeatability tests and the like.
The repeatability (precision) of the method is examined, the coefficient of variation is used for measurement, and the experimental results are shown in table 1.
TABLE 1 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 Table 1:
the intra-batch experimental variation coefficient range of the K417T is 0.25% -2.53%, and the inter-batch experimental variation coefficient range is 0.53% -4.68%;
E484K, the experimental variation coefficient range in batches is 0.32% -2.68%, and the experimental variation coefficient range between batches is 0.63% -3.98%;
the experimental variation coefficient range in the N501Y batch is 0.29-3.25%, and the experimental variation coefficient range between batches is 0.52-4.21%;
the experimental variation coefficients in batches of each mutation detection primer of the method are all less than 3.25 percent, and the experimental variation coefficients in batches are all less than 4.68 percent. Therefore, the method has good repeatability (precision).
Sequence listing
SEQ ID NO.1：5’-CCAGGGCAAACTGGGAC-3’
SEQ ID NO.2：5’-CCAGGGCAAACTGGAAAG-3’
SEQ ID NO.3：5’-CCACCAACCTTAGAATCAAGATTG-3’
SEQ ID NO.4：5’-GCACACCTTGTAATGGTGCTA-3’
SEQ ID NO.5：5’-GCACACCTTGTAATGGTGTTG-3’
SEQ ID NO.6：5’-AGTTGCTGGTGCATGTAGAAG-3’
SEQ ID NO.7：5’-CAATCATATGGTTTCCAACCCAGTT-3’
SEQ ID NO.8：5’-CAATCATATGGTTTCCAACCCACTA-3’
SEQ ID NO.9：5’-CAGTAAGAACACCTGTGCCTGTT-3’
SEQ ID NO.10：5’-CCAGATGATTTTACAGGCTGCGTTATAGCTTGG-3’
SEQ ID NO.11：5’-CCAACCCACTAATGGTGTTGGTTACCAACC-3’
SEQ ID NO.12：5’-CTACATGCACCAGCAACTGTTTGTGGACC-3’
Sequence listing
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<120> qRT-PCR method for identifying novel coronavirus Gamma variant strain
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Claims (2)
1. A qRT-PCR method for identifying a novel coronavirus Gamma variant strain 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 ARMS:
step 2.1, primer design: according to the general principle of primer design, combining nucleotide sequences near the variation sites of the novel coronavirus Indian variety, and designing two pairs of primers for each variation site, wherein the 3' end of an upstream primer is respectively matched with a variation point and a non-variation point, namely a variation upstream primer and a non-variation upstream primer, and the two pairs of upstream primers share a downstream primer;
when the mutation site to be identified is K417T, the nucleotide sequence of the mutation upstream primer is shown in SEQ ID NO. 1: 5'-CCAGGGCAAACTGGGAC-3', the nucleotide sequence of the non-variant upstream primer is shown in SEQ ID NO. 2: 5'-CCAGGGCAAACTGGAAAG-3', the downstream primer is shown in SEQ ID NO. 3: 5'-CCACCAACCTTAGAATCAAGATTG-3', respectively;
when the mutation site to be identified is E484K, the nucleotide sequence of the mutation upstream primer is shown in SEQ ID NO. 4: 5'-GCACACCTTGTAATGGTGCTA-3', the nucleotide sequence of the non-variant upstream primer is shown in SEQ ID NO. 5: 5'-GCACACCTTGTAATGGTGTTG-3', the downstream primer is shown in SEQ ID NO. 6: 5'-AGTTGCTGGTGCATGTAGAAG-3', respectively;
when the mutation site to be identified is N501Y, the nucleotide sequence of the mutation upstream primer is shown in SEQ ID NO. 7: 5'-CAATCATATGGTTTCCAACCCAGTT-3', the nucleotide sequence of the non-variant upstream primer is shown in SEQ ID NO. 8: 5'-CAATCATATGGTTTCCAACCCACTA-3', the downstream primer is shown in SEQ ID NO. 9: 5'-CAGTAAGAACACCTGTGCCTGTT-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, wherein two pairs of primers of each mutation site share one TaqMan probe;
when the mutation site to be identified is K417T, the nucleotide sequence of the TaqMan probe is shown in SEQ ID NO. 10: 5'-CCAGATGATTTTACAGGCTGCGTTATAGCTTGG-3', respectively;
when the mutation site to be identified is E484K, the nucleotide sequence of the TaqMan probe is shown in SEQ ID NO. 11: 5'-CCAACCCACTAATGGTGTTGGTTACCAACC-3', respectively;
when the mutation site to be identified is N501Y, the nucleotide sequence of the TaqMan probe is shown in SEQ ID NO. 12: 5'-CTACATGCACCAGCAACTGTTTGTGGACC-3', respectively;
step 3, performing qRT-PCR experiment based on ARMS;
and 4, interpretation of results:
step 4.1, judging that the mutation occurs in the detection sample when the occurrence time of the amplification curve of the mutation primer system is more than 2 cycles earlier than that of the non-mutation primer system;
and 4.2, judging that no variation occurs in the detection sample when the occurrence time of the amplification curve of the non-variation primer system is more than 2 cycles earlier than that of the variation primer system.
2. The qRT-PCR method for identifying novel coronavirus Gamma variant strain as claimed in claim 1, wherein the qRT-PCR experiment based on ARMS 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, wherein the reaction system 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, 7.5 mu L of sterile RNase-free water and 2 mu L of RNA template, the detection of each mutation site consists of two reaction systems, one contains a mutation upstream primer, the other contains a non-mutation downstream primer, and the other components are the same;
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, 15 seconds to 60 deg.C, 30 seconds.
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