CN112662808A

CN112662808A - Novel coronavirus COVID-19 nucleic acid detection kit and detection method thereof

Info

Publication number: CN112662808A
Application number: CN202011388378.5A
Authority: CN
Inventors: 魏云海; 董海燕; 杨海兵; 邵婕; 金玫华; 徐德顺; 纪蕾
Original assignee: Huzhou Center For Disease Control & Prevention; Huzhou Central Hospital
Current assignee: Huzhou Center For Disease Control & Prevention; Huzhou Central Hospital
Priority date: 2020-12-02
Filing date: 2020-12-02
Publication date: 2021-04-16

Abstract

The invention relates to the technical field of virus detection, in particular to a novel coronavirus COVID-19 nucleic acid detection kit and a detection method thereof, wherein the kit comprises a detection primer group and a probe group, and the detection primer group comprises: specific primer pair aiming at virus ORF1ab gene fragment: ORF1 ab-F: as shown in SEQ ID NO:1, ORF1 ab-R: as shown in SEQ ID NO. 2; specific primer pair for virus N gene fragment: N-F: as shown in SEQ ID NO:3, N-R: as shown in SEQ ID NO. 4. The diagnosis of infection by the novel coronavirus can be aided by the detection of ORF1ab and the N gene fragment. Meanwhile, the sensitivity of the detection of the novel coronavirus can be improved by optimizing the primer sequence, the occurrence of false negative condition is avoided, and the omission factor is reduced.

Description

Novel coronavirus COVID-19 nucleic acid detection kit and detection method thereof

Technical Field

The invention relates to the technical field of virus detection, in particular to a novel coronavirus COVID-19 nucleic acid detection kit and a detection method thereof.

Background

The novel coronavirus belongs to a beta coronavirus, is enveloped, has a circular or elliptical particle, is usually polymorphic, has a diameter of 60-140nm, has a genome sequence of single-stranded RNA with 29903bp, and has obvious gene characteristics different from SARSr-CoV and MERSR-CoV. The present research shows that the homology with bat SARS-like coronavirus (bat-SL-CoVZC 45) reaches more than 85%. Based on the current epidemiological investigation, the latent period of the disease is 1 to 14 days, mostly 3 to 7 days, and the disease is mainly manifested by fever, hypodynamia and dry cough, and a few patients are accompanied by symptoms such as nasal obstruction, pharyngalgia, diarrhea and the like. Severe patients often develop dyspnea and/or hypoxemia after one week of onset, and severe patients rapidly develop acute respiratory distress syndrome, septic shock, refractory metabolic acidosis, hemorrhagic blood coagulation dysfunction and the like.

The novel coronavirus (COVID-19) is incorporated as an acute respiratory infectious disease into a second infectious disease prescribed by the infectious disease control Law of the people's republic of China, and is managed according to the first infectious disease. The situation of the global new coronavirus infection pneumonia is severe, the number of confirmed and suspected cases is still increased, the clinical diagnosis and treatment pressure is huge, and the new coronavirus can continuously exist for a long time. Therefore, the development of a novel high-sensitivity detection system for the coronavirus is beneficial to doing novel coronavirus prevention and control work, practically maintains the health and life safety of people and promotes the stability of the economic society.

A detection primer set of a novel coronavirus (COVID-19) is also reported in the beginning of 2020, for example, a novel coronavirus 2019-nCoV real-time fluorescent quantitative PCR detection primer and probe, a kit and a method which are applied in CN111057797A and 1/19/2020 and disclosed in 24/4/2020; for example, the nucleic acid detection kit for the novel coronavirus covi-19 disclosed in publication No. CN111118228A, application date 3/31/2020, and 5/8/2020, and its method of use are not accurate enough in clinical use and are prone to false positives and false negatives.

Disclosure of Invention

The invention aims to provide a novel coronavirus COVID-19 nucleic acid detection kit and a detection method thereof, which at least solve the problems that the detection accuracy is still to be improved and false positive and false negative are easy to occur in the prior art.

To this end, a first object of the invention is: provides a novel coronavirus COVID-19 nucleic acid detection kit, which comprises two pairs of specific primer pairs with the following nucleotide sequences:

specific primer pair aiming at virus ORF1ab gene fragment:

ORF1 ab-F: as shown in SEQ ID NO. 1,

5′-TTTAGTTCCCTTCCATCATATGCA-3′，

ORF1 ab-R: as shown in SEQ ID NO. 2,

5′-ATTCAGATTTAGCCACATTCAAAGA-3′；

specific primer pair for virus N gene fragment:

N-F: as shown in SEQ ID NO. 3,

5′-ATTGCACAATTTGCCCCCAG-3′，

N-R: as shown in SEQ ID NO. 4,

5′-CATCCAATTTGATGGCACCTG-3′。

the novel coronavirus COVID-19 detection primer group provided by the invention at least has the following beneficial effects:

two pairs of specific primers respectively aim at highly conserved regions of ORF1ab gene and N gene of the novel coronavirus, and the detection of ORF1ab and N gene fragments can assist in diagnosing the infection of the novel coronavirus. Meanwhile, the sensitivity of the detection of the novel coronavirus can be improved by optimizing the primer sequence, the occurrence of false positive and false negative conditions is avoided, and a positive sample with the concentration as low as 15copies/mL can be detected according to a standard curve.

The kit also comprises a probe group which comprises the following probes:

probes against the viral ORF1ab gene fragment:

ORF1 ab-P: as shown in SEQ ID NO. 5,

5′-TTTTGCTACTGCTCAAGAAGCTTATGAGC-3′；

probes against the viral N gene fragment:

N-P: as shown in SEQ ID NO. 6,

5′-TTCTTCGGAATGTCGCGCATTG-3′；

preferably, the probes are labeled with a fluorescent reporter group at the 5 'end and a fluorescent quencher group at the 3' end.

In the PCR amplification process, when the probe is complete, the fluorescence quenching group is close to the fluorescence reporter group, so that the fluorescence emitted by the fluorescence reporter gene is absorbed by the quenching group and does not emit a fluorescence signal. And when the primer is extended, the probe combined with the template is cut off by Taq enzyme (5'→ 3' exonuclease activity), the fluorescent reporter group is separated from the fluorescent quenching group to generate a fluorescent signal, and the fluorescent quantitative PCR instrument automatically draws a real-time amplification curve according to the detected fluorescent signal, so that the detection of the novel coronavirus on the nucleic acid level is realized. The detection result can be selected qualitatively or quantitatively, and the qualitative detection can be realized by directly judging whether the corresponding amplification curve exists or not. Drawing a corresponding standard curve according to the Ct values of the standard substances with different concentration gradients, and calculating according to the Ct values of the samples to obtain a quantitative result. In addition, based on the general understanding in the art, the fluorescent reporter groups of different probes should be different so that the amplification of different gene fragments in a single-tube reaction produces fluorescent signals of different wavelength bands for convenient detection.

Further preferably, ORF1ab-P is labeled with a fluorescence reporter FAM at the 5 'end and a fluorescence quencher BHQ1 at the 3' end; the 5 'end of the N-P is marked with a fluorescence reporter group HEX, and the 3' end is marked with a fluorescence quenching group BHQ 1.

Preferably, the detection kit further comprises an internal standard primer pair and an internal standard probe;

wherein, the nucleotide sequence of the internal standard primer pair is as follows:

RNP-F: as shown in SEQ ID NO. 7,

5′- AGATTTGGACCTGCGAGCG-3′，

RNP-R: as shown in SEQ ID NO. 8,

5′-GAGCGGCTGTCTCCACAAGT-3′；

the nucleotide sequence of the internal standard probe is as follows:

RNP-P: as shown in SEQ ID NO. 9,

5′-TTCTGACCTGAAGGCTCTGCGCG-3′。

the internal standard used by the kit is human ribonuclease P (RNase P, RNP), whether qualified patient tissues or secretion samples are obtained or not is judged through the internal standard, the whole experimental process of collection, transportation and extraction of samples to be detected is monitored in a full-scale mode, and false negative of detection results is avoided.

Preferably, the internal standard probe RNP-P is labeled with a fluorescent reporter group at the 5 'end and a fluorescent quencher group at the 3' end.

The fluorescent reporter group of the internal standard probe should also be a different group from the fluorescent reporter group of the detection probe, for example, TEXAS RED is selected and BHQ2 is selected as the fluorescence quenching group, so that fluorescence can be detected in TEXAS RED/ROX channel.

Preferably, the detection kit provided by the invention further comprises a mixed solution of RT-PCR enzyme and buffer solution.

Preferably, the detection kit provided by the invention further comprises a positive control substance and a negative control substance. The positive control should be capable of completely amplifying the target gene segment corresponding to the primer during the amplification process, for example, it can be an inactivated standard strain, a plasmid containing the target gene segment (ORFlab gene segment, N gene segment, RNP gene segment) corresponding to the viral primer probe, etc. The negative control is water for dissolving the template RNA, and may be DEPC water, for example.

Preferably, the detection kit provided by the invention further comprises a nucleic acid extraction reagent. The extraction of the virus nucleic acid in the sample is completed through the corresponding nucleic acid extraction reagent so as to facilitate the subsequent amplification detection procedure.

The second purpose of the invention is to provide a novel detection method of coronavirus COVID-19, which uses the detection kit.

The detection method comprises the following steps:

1. extracting total RNA of a sample to be detected;

2. carrying out reverse transcription on the total RNA of the sample, and taking the total RNA as a template, and carrying out amplification by using the detection primer group and the probe group;

3. analyzing the amplification product, and judging whether the sample to be detected contains the novel coronavirus or not according to whether an amplification curve appears or not;

according to the detection method of some embodiments of the present invention, the procedure of the PCR amplification reaction is 50 ℃ for 10 min; 95 ℃ for 10 s; 95 ℃, 5s, 60 ℃, 30s, 40 cycles.

The real-time fluorescence RT-PCR method only needs about 1 hour and 10 minutes from the extraction of nucleic acid to the completion of detection, is convenient to operate and high in sensitivity, and provides a quick and effective laboratory detection method for the diagnosis of epidemic situations.

Two pairs of specific primers respectively aim at highly conserved regions of ORF1ab gene and N gene of the novel coronavirus, and the detection of ORF1ab and N gene fragments can assist in diagnosing the infection of the novel coronavirus. Meanwhile, the sensitivity of the detection of the novel coronavirus can be improved by optimizing the primer sequence, the occurrence of false negative condition is avoided, and the omission factor is reduced.

The detection method using the kit can be used for auxiliary diagnosis of the novel coronavirus, rapidly obtains epidemiological information about the outbreak of the novel coronavirus, provides reliable technical support for prevention and control of the outbreak of the novel coronavirus, and has important significance in dealing with the outbreak of the unknown novel coronavirus in the future.

Drawings

FIG. 1: the amplification result of the novel coronavirus multiplex PCR detection kit of example 4 of the invention.

FIG. 2: a standard curve and an amplification curve of the same sample at different dilution concentrations, and FIG. 2-1 is a standard curve prepared based on the amplification curve of FIG. 2-2The concentration of positive plasmid was 4ug/ml, respectively, corresponding to a copy number of 1 × 10¹⁰copy/ml, and the ratio of C1-C7 after dilution is 1 x 10^2~8copy/ml. By Ct<40 is a negative judgment standard, and the lower detection limit of the development kit is 15 copy/ml.

FIG. 3: RNA is extracted from the stock solution of the positive sample D, the RNA is respectively detected by a research and development kit, a randomly drawn kit A (figure 3-1), a kit B (figure 3-2) and a kit C (figure 3-3) in the market, amplification conditions are respectively amplified according to the specifications of the kits A, B and C, all the kits detect that the sample D is positive, the Ct value of the kit is smaller than that of the randomly drawn kit, and the sensitivity is higher.

FIG. 4-1: the positive sample D stock solution is diluted by 8 times to extract RNA as a template, a research and development kit, a kit A (figure 4-1) randomly drawn in the market and a PCR reaction system and an amplification condition are respectively used as reaction conditions provided by the instruction of the kit A, and the highest CT value of the sensitivity of the research and development kit is 36.2, and the positive sample is regarded as a positive sample. And the CT value of the kit A is up to 38.3, and the kit A is judged to be a suspicious sample according to the kit instruction.

FIG. 4-2: the positive sample D stock solution is diluted and extracted to obtain RNA as a template, a research and development kit, a kit B (figure 4-2) randomly drawn in the market, a PCR reaction system and an amplification condition are reaction conditions provided by the specification of the kit B, and the highest sensitivity CT value of the research and development kit is 36.4, and the positive sample D is regarded as a positive sample. And the CT value of the kit B exceeds 40, and the kit B is judged to be a negative sample according to the kit instruction, so that false negative is caused.

FIGS. 4-3: the positive sample D stock solution is diluted by 8 times to extract RNA as a template, a research and development kit, a kit C (figure 4-3) randomly drawn in the market and a PCR reaction system and an amplification condition are respectively used as reaction conditions provided by the instruction of the kit C, and the highest sensitivity CT value of the research and development kit is 38.2, and the research and development kit is regarded as a suspicious sample. And the CT value of the kit C exceeds 40, and the kit C is judged to be a negative sample according to the kit instruction, so that false negative is caused.

Detailed Description

The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention.

Example 1

The embodiment provides a primer group and a probe of a novel coronavirus, based on a Taqman fluorescent probe technology, corresponding specificity detection primers and Taqman probes are designed by using highly conserved regions of ORF1ab gene and N gene of the novel coronavirus, and meanwhile, a corresponding internal standard primer pair and an internal standard probe are designed by using human RNP as an internal standard. The details are shown in table 1 below:

TABLE 1 primer and Probe information

Verification of sensitivity of primer set and Probe of example 1

The detection method comprises the following steps:

a positive plasmid template is shared by the ORF1ab gene fragment and the N gene fragment, and the PCR reaction system and the PCR program are shown in Table 2:

TABLE 2 RT-PCR reaction solution System

Negative quality control as template, adding 5 μ L into the above reaction solution system, and performing multiple fluorescence PCR amplification reaction at 50 deg.C for 10min (if the step is synchronous with the sample, it is not removed, and if only positive plasmid amplification is performed, it can be omitted); 95 ℃ for 10 s; 95 ℃, 5s, 60 ℃, 30s, 40 cycles.

Example 2

The embodiment provides a novel coronavirus detection kit, which comprises a mixed solution of RT-PCR enzyme and a buffer solution, a mixed solution of a primer and a probe, a positive control substance and a negative control substance.

The mixture of RT-PCR enzyme and buffer is a commercial reagent.

The primer and probe mixture was the primer and probe of example 1;

the positive control is a plasmid solution of primer targeting fragments of novel coronavirus ORF1ab gene and N gene and internal standard gene with pUC57 as plasmid vector.

The negative control was DEPC water.

Example 3

The embodiment provides a method for detecting a novel coronavirus, which comprises the following steps:

1. extraction of nucleic acids

Obtaining a sample to be detected, inactivating (heating at 56 ℃ for 30min), and extracting according to a method for extracting the commercialized kit.

The sample to be tested may be any one of a pharyngeal swab, a nasopharyngeal secretion, an alveolar lavage fluid, a sputum, a serum, and a plasma.

The extracted nucleic acids may be RNA and/or DNA.

PCR amplification

The reagents were removed, thawed at room temperature or on ice and shaken well, centrifuged briefly. The reaction number N = (number of samples + negative quality control + positive quality control + 1) × the number of multiple wells was calculated. An RT-PCR reaction solution system was prepared according to Table 3:

TABLE 3 RT-PCR reaction solution System

The mixture of RT-PCR enzyme and buffer was the mixture of RT-PCR enzyme and buffer in example 2.

Adding 5 mu L of RNA extracted in the step 1 as a template into the reaction solution system, and performing multiple fluorescence PCR amplification reaction on an ABI 7500 instrument, wherein the amplification reaction procedure is as follows, and the temperature is 50 ℃ for 10 min; 95 ℃ for 10 s; 95 ℃, 5s, 60 ℃, 30s, 40 cycles.

3. Result judgment

Setting a baseline and a threshold line, wherein the baseline is generally 3-15 cycles, and the threshold line is the highest point just exceeding the negative control.

The positive control substance needs to satisfy the condition that FAM, HEX and TEXAS RED channels have obvious S-type amplification curves; the negative control needs to satisfy no amplification curve; and the result of the sample to be detected can be analyzed by meeting the conditions.

If the two channels of FAM and HEX both detect obvious S-type amplification curves, the sample to be detected contains the novel coronavirus, and only one channel detects the obvious S-type amplification curve and needs to be detected again.

FIG. 1 shows the amplification results of the novel coronavirus multiplex PCR detection kit of example 3 of the present invention. As can be seen, the FAM, HEX and TEXAS RED channels have distinct sigmoid amplification curves, indicating that the biological samples are of human origin and contain the novel coronavirus.

Example 4

In this example, the detection method of example 3 was used, and the same template was diluted to different concentrations for detection, and the amplification curve of the result was shown in FIG. 2-2. And drawing a standard curve according to the amplification curve as shown in the attached figure 2-1, wherein the result shows that the lower detection limit of the kit can reach 15copy/ml by taking Ct <40 as a judgment standard.

Example 5

RNA is extracted from the same positive sample stock solution, the kit developed by the invention, the kit A, the kit B and the kit C randomly drawn in the market are respectively used for detection, and the amplification graph is shown as the attached figure 3. The Ct value of the kit is smaller than that of the randomly extracted kit, and the sensitivity is higher.

The positive sample stock solution is diluted 8 times to extract RNA, and the RNA is respectively extracted by using a research and development kit, a kit A (Shanghai Jieno biotechnology limited, 2019 novel coronavirus ORF1a/E/N gene three-target detection kit, the product number GZ-TRM 25) randomly drawn by the market, a kit B (Beijing Zhuo Corchorsheng biotechnology limited, novel coronavirus 2019-nCoVORFab/B, 7712 RC-50T), a kit C (Shanghai Jiang biotechnology limited, novel coronavirus 2019-NcoV nucleic acid detection kit, Z-RR-0479-02-50), a PCR reaction system and reaction conditions are operated according to the kit specification, and fluorescence is uniformly collected for data analysis. The highest CT value of the sensitivity of the development kit is 36.2, the kit A can detect that the CT value is as high as 38.3, other randomly drawn two kits cannot detect false negative, and the amplification graphs are shown in the attached figures 4-1 to 4-3.

Example 6

This example was compared with CDC published primer and probe combinations as controls for sensitivity.

Table 5 comparison of the results of the tests performed on the two batches using different methods.

As can be seen from Table 5, the Ct value of ORF1ab gene amplified in this item is 1.01-1.04 smaller than that of CDC recommended probe primer combination, i.e., the sensitivity is improved by 2.01-2.06 times of CDC, and the Ct value of N gene amplified is 1.32-1.49 smaller than that of CDC recommended probe primer combination, i.e., the sensitivity is improved by 2.50-2.81 times of CDC.

Example 7

In this example, 2 different samples were simultaneously detected in comparison with a commercially available kit (code A), and the samples were amplified by detection according to the kit instructions and the computer program. As can be seen from Table 4, the Ct value of ORF1ab gene amplified by this item is 1.12-1.28 smaller than that of brand A, i.e. the sensitivity is 2.17-2.43 times that of brand A, and the Ct value of N gene amplified is 2.20-2.69 smaller than that of brand A, i.e. the sensitivity is 4.59-6.45 times that of brand A. This shows that the detection kit provided by the embodiment of the invention has higher detection sensitivity than a control group and good reagent amplification sample effect.

Table 4. kits for two batches of samples the assay results were compared laterally.

The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present invention. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.

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Claims

1. The novel coronavirus COVID-19 nucleic acid detection kit is characterized by comprising a detection primer group and a probe group, wherein the detection primer group comprises: specific primer pair aiming at virus ORF1ab gene fragment:

ORF1 ab-F: as shown in SEQ ID NO. 1,

ORF1 ab-R: as shown in SEQ ID NO. 2;

specific primer pair for virus N gene fragment:

N-F: as shown in SEQ ID NO. 3,

N-R: as shown in SEQ ID NO. 4.

2. The novel coronavirus COVID-19 nucleic acid detection kit according to claim 1, wherein the probe set comprises two probes having the following nucleotide sequences:

probes against the viral ORF1ab gene fragment:

ORF1 ab-P: as shown in SEQ ID NO. 5;

probes against the viral N gene fragment:

N-P: shown as SEQ ID NO. 6.

3. The kit for detecting the CoVID-19 nucleic acid of claim 1, wherein the probe is labeled with a fluorescent reporter group at the 5 'end and a fluorescent quencher group at the 3' end.

4. The novel coronavirus COVID-19 nucleic acid detection kit according to claim 1, wherein the 5 'end of the probe of the ORF1ab-P gene fragment is labeled with a fluorescence reporter FAM, and the 3' end is labeled with a fluorescence quencher BHQ 1; the 5 'end of the probe of the N-P gene fragment is marked with a fluorescence reporter group HEX, and the 3' end is marked with a fluorescence quenching group BHQ 1.

5. The kit for detecting the novel coronavirus COVID-19 nucleic acid according to claim 1, further comprising an internal standard primer pair; wherein, the nucleotide sequence of the internal standard primer pair is as follows:

RNP-F: as shown in SEQ ID NO. 7,

RNP-R: as shown in SEQ ID NO. 8.

6. The kit for detecting the nucleic acid of the novel coronavirus COVID-19 according to claim 1, further comprising an internal standard probe,

the nucleotide sequence of the internal standard probe is as follows:

RNP-P: shown as SEQ ID NO. 9.

7. The kit for detecting the CoVID-19 nucleic acid of the coronavirus of claim 1, wherein the RNP-P is labeled with a fluorescent reporter group at the 5 'end and a fluorescent quencher group at the 3' end.

8. The kit for detecting the CoVID-19 nucleic acid of the novel coronavirus according to claim 1, wherein the 5 'end of RNP-P is labeled with a fluorescent reporter group TEXAS RED, and the 3' end is labeled with a fluorescent quencher group BHQ 2.

9. The kit for detecting the COVID-19 nucleic acid of claim 1, further comprising RT-PCR enzyme mixture and RT-PCR buffer.

10. The test kit of claim 5, further comprising a positive control and a negative control.