CN111549175A - Novel multi-fluorescence RT-PCR detection reagent for detecting port input 2019 coronavirus - Google Patents
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Abstract
The invention discloses a multiple fluorescence PCR primer, a probe and a kit for identifying 2019 novel coronaviruses, wherein the primer is shown as a sequence from SEQ ID No.1 to SEQ ID No.10, and the probe is shown as a sequence from SEQ ID No.11 to SEQ ID No. 15.
Description
Technical Field
The invention relates to a novel multi-fluorescence RT-PCR detection kit and oligonucleotide for detecting port input 2019, and belongs to the field of inspection and quarantine.
Background
2019A novel coronavirus (Severe acid respiratory syndrome coronavirus 2, SARS-Cov-2) is a single-stranded positive-strand RNA virus of the genus Beta of coronavirus with the diameter of 60-140nm, is a newly discovered member of the genus Beta of coronavirus, and diagnosis cases have been reported in 200 countries and regions at present. After a person is infected with the 2019 novel coronavirus, the common signs of the person are respiratory symptoms, fever, cough, shortness of breath, dyspnea and the like. In more severe cases, the infection may lead to clinical manifestations of different symptoms of pneumonia, severe acute respiratory syndrome, renal failure, and even death.
SARS-Cov-2 prevention and control is in the key stage, and nucleic acid detection is used as the main means for accurate diagnosis and plays an important role in rapid diagnosis, treatment effect evaluation and epidemic prevention and control. By 26 days 3 months, 23 SARS-Cov-2 detection products, 15 SARS-Cov-2 nucleic acid detection reagents and 8 antibody detection reagents, have been approved by the relevant departments. However, because the novel coronavirus pneumonia is a sudden epidemic situation, the time for applying the nucleic acid detection method to clinic is short, sufficient performance verification and clinical application evaluation are not carried out yet, and a false negative phenomenon that the nucleic acid detection result is inconsistent with clinical diagnosis occurs in sample detection. Meanwhile, SARS-Cov-2 is a single-stranded positive-strand RNA virus, and the nucleic acid sequence of the RNA virus is very easy to mutate, so that after SARS-Cov-2 is popular in a large scale in the population all over the world and the virus is likely to rapidly evolve, the problems of missed detection and low specificity and other technical defects exist in the existing diagnostic kit, and the problems of optimizing the current detection means and improving the nucleic acid detection sensitivity of foreign personnel imported from the port of China are urgently needed to be solved.
Disclosure of Invention
The first technical problem to be solved by the invention is to provide a group of oligonucleotides with strong specificity and high sensitivity for detecting 2019 novel coronavirus, which comprises a primer and a probe.
The second technical problem to be solved by the invention is to provide a multiple fluorescence RT-PCR detection kit for specifically, sensitively and efficiently detecting 2019 novel coronavirus.
The third technical problem to be solved by the invention is to provide a multiple fluorescence RT-PCR detection method for detecting 2019 novel coronavirus.
In order to solve the first technical problem, the invention adopts the following technical scheme:
a group of oligonucleotides for detecting 2019 novel coronaviruses is oligonucleotides shown in sequence tables SEQ ID No.1 to SEQ ID number 15, wherein the sequences SEQ ID No.1 to SEQ ID No.10 are respectively a sense primer and an antisense primer for detecting 2019 novel coronaviruses, and the sequences SEQ ID No.11 to SEQ ID No.15 are fluorescent probes for detecting 2019 novel coronaviruses, and the oligonucleotides specifically comprise:
primer RdRP _ SARSr-FP 1: 5'-ATGGCCTCACTTGTTCTTGC-3', (SEQ ID No. 1);
primer RdRP _ SARSr-RP 1: 5'-CGCCACACATGACCATTTCA-3', (SEQ ID No. 2);
probe RdRP _ SARSr-P1: 5'-ACGTGTTGTAGCTTGTCACACCGT-3', (SEQ ID No. 11); the 5 'end of the probe is marked with a report fluorescent group FAM, and the 3' end of the probe is marked with a quenching fluorescent group BHQ 1. The length of the target fragment amplified by the primer and the probe is 115 bp.
Primer RdRP _ SARSr-FP 2: 5'-CCTCATCAGGAGATGCCACA-3', (SEQ ID No. 3);
primer RdRP _ SARSr-RP 2: 5'-GCGGACATACTTATCGGCAA-3', (SEQ ID No. 4);
probe RdRP _ SARSr-P2: 5'-TGTCAAGCTGTCACGGCCAATGT-3', (SEQ ID No. 12); the 5 'end of the probe is marked with a reporter fluorescent group CY5, and the 3' end is marked with a quenching fluorescent group BHQ 2. The length of the target fragment amplified by the primer and the probe is 125 bp.
Primer N _ SARSr-FP 1: 5'-GTTCACCGCTCTCACTCAAC-3', (SEQ ID No. 5);
primer N _ SARSr-RP 1: 5'-GTTCACCGCTCTCACTCAAC-3', (SEQ ID No. 6);
probe N _ SARSr-P1: 5'-CCCTCGAGGACAAGGCGTTCCA-3', (SEQ ID No. 13); the 5 'end of the probe is marked with a reporter fluorophore HEX, and the 3' end is marked with a quenching fluorophore BHQ 1. The length of the target fragment amplified by the primer and the probe is 125 bp.
Primer N _ SARSr-FP 2: 5'-GCTGCTGCTTGACAGATTGA-3', (SEQ ID No. 7);
primer N _ SARSr-RP 2: 5'-GCCTCRGCMGCAGATWTCTT-3', (SEQ ID No. 8);
probe N _ SARSr-P2: 5'-AGGCCAACAACAACAAGGCCA-3', (SEQ ID No. 13); the 5 'end of the probe is marked with a reporter fluorophore VIC, and the 3' end of the probe is marked with a quenching fluorophore BHQ 2. The length of the target fragment amplified by the primer and the probe is 99 bp.
Primer S _ SARSr-FP 1: 5'-ATGTCCTTCCCTCAGTCAGC-3', (SEQ ID No. 9);
primer S _ SARSr-RP 1: 5'-AAATGGCAGGAGCAGTTGTG-3', (SEQ ID No. 10);
probe S _ SARSr-P1: 5'-TGCATGTGACTTATGTCCCTGCACA-3', (SEQ ID No. 15); the 5 'end of the probe is marked with a reporter fluorescent group Texas Red, and the 3' end is marked with a quenching fluorescent group BHQ 1. The length of the target fragment amplified by the primer and the probe is 97 bp.
In order to solve the second technical problem, the invention adopts the following technical scheme:
the invention provides a qPCR detection kit for detecting novel coronavirus, which comprises the following components:
(1) the qPCR reaction solution comprises a sense primer shown in a sequence table SEQ ID No.1/3/5/7/9, an antisense primer shown in a sequence table SEQ ID No.2/4/6/8/10 and a fluorescent probe shown in a sequence table SEQ ID No.11/12/13/14/15, wherein the 5 'end of the probe is marked with a report fluorescent group FAM, and the 3' end of the probe is marked with a quenching fluorescent group BHQ 1;
(2) negative control: adopting a flocking sterile long cotton swab to quickly wipe secretions of the palatine arches, the pharynx and the tonsils on two sides of a novel coronavirus 2019 person without infection, inserting the cotton swab into a culture tube, vibrating the culture tube for 1 minute, taking 200 mu L of pharynx swab culture solution, extracting RNA, and dissolving in ddH2O；
(3) Positive control: quickly wiping secretions of a palatine arch, a pharynx and a tonsil of a 2019 novel coronavirus infected patient on two sides by adopting a flocking sterile long cotton swab, inserting the cotton swab into a culture tube, shaking the culture tube for 1 minute, taking 200 mu L of pharynx swab culture solution, extracting RNA, and dissolving in DEPC water; .
Further, the RT-qPCR reaction solution also comprises reagents and enzymes required by the conventional qPCR reaction, and preferably, the qPCR reaction solution also comprises 4 × TaqManTMFast 1-Step Master Mix from ABI, ddH2O was purchased from TaKaRa.
In order to solve the third technical problem, the invention also provides a qPCR detection method for detecting 2019 novel coronavirus, which comprises the following steps:
(1) extracting sample RNA;
taking throat swab carrying 2019 novel coronavirus, extracting RNA by using RNA extraction kit (QIAamp Viral RNA MiniKit, purchased from Qiagen company), or extracting RNA by using a method known in the prior art;
(2) qPCR amplification of extracted sample RNA:
TABLE 1qPCR reaction System
Reaction conditions are as follows:
10min 1Cycle at 50 ℃; 5min 1Cycle at 95 ℃; then 95 ℃ for 5s and 60 ℃ for 30s for 45 cycles.
(3) And (4) judging a result:
setting the analysis conditions of the results: the threshold setting rule is that the threshold line just exceeds the highest point of the normal negative control amplification curve. CTThe value is less than or equal to 35, and the obvious amplification curve is positive, which indicates that SARS-Cov-2 exists in the sample; is free of CTValues, and no amplification curve is negative, indicating that SARS-Cov-2 is absent from the sample.
Aiming at the current efficient method for detecting high sensitivity and specificity of the novel needleless entry 2019 coronavirus, the invention provides a multiple qPCR detection method for detecting the novel 2019 coronavirus, which is simple, rapid, sensitive and accurate in operation, and can achieve the aim of accurately quantifying SARS-Cov-2 in a sample to be detected.
The invention has the beneficial effects that:
1. the primer and the probe provided by the invention can be used for qualitative and quantitative analysis of 2019 novel coronavirus, and have important significance for the research on the aspects of judging the reproduction rule and the reproduction level of SARS-Cov-2 in a human body, whether the SARS-Cov-2 is eliminated after medication and the like;
2. the invention plays an important role in large-scale quarantine, epidemiological investigation and early diagnosis of the 2019 novel coronavirus.
Drawings
Figure 1 is a 2019 novel coronavirus primer and probe specific amplification curve.
FIG. 2 is a multiplex qPCR amplification curve for 6 samples tested.
Detailed Description
The invention will now be further illustrated by reference to specific examples. However, these examples are only illustrative and not intended to limit the scope of the present invention.
The experimental procedures in the following examples are conventional unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 design of primers and probes specific for detection 2019 of a novel coronavirus
SARS-Cov-2 whole genome sequence of all parts of the world is obtained from GenBank and GISAID, 3356 pieces of data base are marked as genome, each motif is aligned to 3148 sequences respectively aligned with genome, parameter value is 0.01 (99.99% reliability), sequence consistency is more than 90%, except that whole genome sequence with complete gene fragment less than 29000bp and complete identity, sequence comes from 40 countries and regions, MEGA X software evolution analysis, 413 genome sequences are obtained. Respectively obtaining the total length of 413 RdRP genes, N genes and S genes of a novel 2019 coronavirus whole genome, respectively obtaining the conserved gene sequences of SARS-Cov-2RdRP genes, N genes and S genes, designing a primer and a TaqMan probe by using software PrimerExpress 3.0 according to the general principle of primer and probe design, comparing the designed primer and probe on NCBI, and finally determining 5 pairs of primers and 5 probes by conventional screening, wherein the sequences are as follows: primers SEQ ID No.1 to SEQ ID No. 10); probes SEQ ID No.11 to SEQ ID number 15). The 5 'end of the probe contains FAM, CY5, HEX, VIC and TEXAS RED reporter fluorescent dyes respectively, and the 3' end of the probe contains a quenching fluorescent dye BHQ 1. All primers and probes were synthesized by Shanghai Huada Biotech.
Example 2 composition of multiplex RT-qPCR assay kit for detecting 2019 novel coronaviruses
(1) The qPCR reaction solution comprises a sense primer shown in a sequence table SEQ ID No.1/3/5/7/9, an antisense primer shown in a sequence table SEQ ID No.2/4/6/8/10 and a fluorescent probe shown in sequence tables SEQ ID No.11-15, wherein a 5 'end of the probe is marked with a reporter fluorescent group containing FAM, CY5, HEX, VIC and TEXAS RED reporter fluorescent dyes, and a 3' end is marked with a quenching fluorescent group BHQ 1;
(2) negative control: negative control: adopting a flocking sterile long cotton swab to quickly wipe secretions of palatine arches, pharynx and tonsil on two sides of a novel coronavirus-free person 2019, inserting the cotton swab into a culture tube, vibrating the culture tube for 1 minute, taking 200 mu L of pharynx swab culture solution, extracting RNA, and dissolving in ddH2O；
Further, the qPCR reaction solution also comprises 4 × TaqManTMFast 1-Step Master Mix and ddH2O。
Example 3 specific assay for multiplex RT-qPCR detection 2019 of novel coronaviruses
The specific process comprises the following steps:
first, the source of the sample
2019 novel coronavirus (Severe atmospheric respiratory syndrome coronavirus 2, SARS-Cov-2), Severe atmospheric respiratory syndrome coronavirus (Severe atmospheric respiratory syndrome coronavirus, SARSr-CoV) and Middle East respiratory syndrome coronavirus (Middle East respiratory syndrome coronavirus, MERSR-CoV) were preserved by Beijing customs laboratories.
Second, extraction of sample RNA
Extracting total plant DNA from the sample obtained in the step one by using a DNA extraction kit (purchased from Qiagen company), wherein the specific operation is shown in the kit instruction; (1) extracting sample RNA;
the sample carrying the 2019 novel coronavirus is taken, and RNA is extracted by a RNA extraction kit (QIAamp Viral RNA MiniKit, purchased from Qiagen company), or the RNA can be extracted by a method known in the prior art;
third, specificity detection test
Taking the sample RNA obtained in the first step and the second step as a template, namely: RNA of SARS-Cov-2, SARSr-CoV and MERSR-CoV, water as blank control, and qPCR reaction.
TABLE 1qPCR reaction System
Reaction conditions are as follows:
10min 1Cycle at 50 ℃; 5min 1Cycle at 95 ℃; then 95 ℃ for 5s and 60 ℃ for 30s for 45 cycles.
As shown in FIG. 1, the designed primers and probes have strong specificity, and only SARS-Cov-2, but not SARSr-CoV and MERSR-CoV, can be detected.
Example 4 multiplex RT-qPCR detection 2019 novel coronavirus Beijing port personnel entry detection example
Total RNA is extracted from 6 passengers from different countries entering the entrance of Beijing port according to the detection method of the invention, and then RT-qPCR reaction is carried out. The amplification curve is shown in FIG. 2.
It should be understood that the above-described embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. Not all embodiments are exhaustive. All obvious changes and modifications which are obvious to the technical scheme of the invention are covered by the protection scope of the invention.
Sequence listing
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Claims (6)
1. A novel multi-fluorescence RT-PCR detection reagent for detecting port input 2019 coronavirus is characterized in that the oligonucleotide is shown as SEQ ID No.1 to SEQ ID No.15 in a sequence table; wherein the sequences SEQ ID No.1/3/5/7/9 and SEQ ID No.2/4/6/8/10 are respectively a sense primer and an antisense primer for detecting 2019 novel coronavirus, and the sequence SEQ ID No.11/12/13/14/15 is a fluorescent probe for detecting 2019 novel coronavirus.
2. The oligonucleotide for detecting 2019 novel coronaviruses according to claim 1, wherein the fluorescent probe sequence SEQ ID No.11/12/13/14/15 is labeled at the 5 'end with a reporter fluorophore FAM, CY5, HEX, VIC, TEXASRED and at the 3' end with a quencher fluorophore BHQ 1.
3. Use of the oligonucleotide for detecting 2019 novel coronavirus according to claim 1 or 2 in preparation of a 2019 novel coronavirus detection kit.
4. A qPCR detection kit for 2019 novel coronavirus, which is characterized by comprising the following components:
(1) a qPCR reaction solution, comprising: a sense primer shown in a sequence table SEQ ID No.1/3/5/7/9, an antisense primer shown in a sequence table SEQ ID No.2/4/6/8/10 and a fluorescent probe shown in a sequence table SEQ ID No.11/12/13/14/15, wherein the 5 'end of the fluorescent probe is marked with report fluorescent groups FAM, CY5, HEX, VIC and TEXAS RED, and the 3' end of the fluorescent probe is marked with a quenching fluorescent group BHQ 1;
(2) negative control;
(3) and (4) positive control.
5. The kit according to claim 4, wherein the qPCR reaction solution further comprises 4 × TaqManTMFast 1-Step Master Mix and ddH2O。
6. A qPCR detection method of 2019 novel coronavirus, which is characterized by comprising the following steps:
(1) extracting sample RNA;
(2) performing qPCR amplification on the extracted sample RNA; the fluorescent probe comprises a sense primer shown in a sequence table SEQ ID No.1/3/5/7/9, an antisense primer shown in a sequence table SEQ ID No.2/4/6/8/10 and a fluorescent probe shown in a sequence table SEQ ID No.11/12/13/14/15, wherein the 5 'end of the fluorescent probe is marked with a report fluorescent group FAM, CY5, HEX, VIC and TEXAS RED, and the 3' end of the fluorescent probe is marked with a quenching fluorescent group BHQ 1;
(3) and (4) judging the result according to the fluorescence intensity of the qPCR reaction system.
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| PCT/CN2020/084399 WO2021203448A1 (en) | 2020-04-07 | 2020-04-13 | Multiplex fluorescent rt-pcr detection reagent for detecting coastal input severe acute respiratory syndrome coronavirus 2 |
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| KR102421844B1 (en) | 2021-04-16 | 2022-07-15 | 한국생명공학연구원 | Novel Marker for diagnosis of Sars coronavirus2 infection and use thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| KR102421844B1 (en) | 2021-04-16 | 2022-07-15 | 한국생명공학연구원 | Novel Marker for diagnosis of Sars coronavirus2 infection and use thereof |
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