CN113186346A - Novel coronavirus nucleic acid PCR-colloidal gold immunochromatography detection kit - Google Patents
Novel coronavirus nucleic acid PCR-colloidal gold immunochromatography detection kit Download PDFInfo
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Abstract
The invention discloses a novel coronavirus nucleic acid PCR-colloidal gold immunochromatography detection kit, which comprises: amplification reaction solution, CoV detection solution, positive control substance and negative control substance; the CoV detection solution comprises specific marker primers aiming at the open reading frame 1ab of the new coronavirus and the gene encoding the nucleocapsid protein N, specific marker primers of the internal reference gene RNase P and RNase Free ddH2And O. The kit is prepared by extracting nucleic acid from collected cell sample, adding into reverse transcriptase andTaqunder the action of DNA polymerase, specific fragments are amplified, and then the colloidal gold immunochromatography is utilized to realize the detection of the nucleic acid of the new coronavirus, in addition, the kit adopts reference gene RNase P to carry out tracking quality control on the processes of sample collection, nucleic acid extraction and PCR amplification, thereby ensuring the whole experimental processAre all within a controllable range.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a novel coronavirus (COVID-19) nucleic acid colloidal gold immunochromatography detection kit.
Background
Coronaviruses are a large family of viruses known to cause the common cold and more serious diseases such as Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS). The novel coronavirus is a new strain of coronavirus that has not been previously discovered in humans.
The main transmission path of the novel coronavirus is respiratory droplet transmission and contact transmission, and the transmission paths of aerosol, feces, mouth and the like are yet to be further defined. Epidemiological investigation shows that many cases can be traced to the close contact with the diagnosed cases.
After people are infected with 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 can lead to pneumonia, severe acute respiratory syndrome, renal failure, and even death. The viral lethality rate is about 2% to 4%, but this is a very early percentage that may change as more information is obtained. At the same time, this does not mean that it is not serious, just that a person infected with a virus does not necessarily face the most serious consequences. At present, no specific treatment method is available for diseases caused by the novel coronavirus.
The current laboratory detection scheme of the new coronavirus infection is mainly a fluorescent quantitative PCR method. The method has the advantages of high sensitivity, short time consumption and relatively high flux, but the detection process of the method depends on an expensive qPCR instrument, and the requirement of a primary hospital is difficult to meet.
The nucleic acid colloidal gold immunochromatography technology is characterized in that specific antigens or antibodies are fixed on an NC membrane in a strip shape to form a detection line (T line), an internal reference line (R line) and a quality control line (C line), and a colloidal gold labeled reagent (antibodies or monoclonal antibodies) is adsorbed on a binding pad. After a sample to be detected is extracted by conventional nucleic acid, PCR amplification is carried out by adopting a primer with a specific mark at the 5' end, an amplification product is directly added on a sample pad at one end of a test strip, the amplification product moves forwards through capillary action and interacts with a colloidal gold labeling reagent on a combination pad, and when the amplification product moves to a region of a fixed antigen or antibody, a combination of an object to be detected and the colloidal gold labeling reagent is specifically combined with the combination to be intercepted and gathered on a detection band to form a visual color development result. The method is widely applied to antigen-antibody detection, but is rarely used in the field of nucleic acid detection.
In the whole experiment process, pollution or other interference factors may appear by sample nucleic acid extraction and PCR amplification, and the traditional colloidal gold only has a detection line and a quality control line, cannot be used for setting reference gene detection, cannot effectively monitor the whole nucleic acid detection process, and may cause false negative results and interference judgment.
The invention aims to establish a kit which can simply, conveniently, quickly, accurately and high-flux detect the infection condition of the new coronavirus by utilizing a PCR (polymerase chain reaction) technology and a colloidal gold immunochromatography technology. The reagent kit is internally provided with an internal reference gene for whole-process quality control, so that false negative result interference can be avoided.
Disclosure of Invention
Aiming at the technical problems in the prior art, the invention provides a novel coronavirus nucleic acid PCR-colloidal gold immunochromatography detection kit, which can overcome the defects in the prior art.
In order to achieve the technical purpose, the technical scheme of the invention is realized as follows:
a novel coronavirus nucleic acid PCR-colloidal gold immunochromatography detection kit comprises: amplification reaction solution, CoV detection solution, positive control substance and negative control substance;
the CoV detection solution comprises specific marker primers aiming at the open reading frame 1ab of the new coronavirus and the gene encoding the nucleocapsid protein N, specific marker primers of the internal reference gene RNase P and RNase Free ddH2O；
The sequence of the specific labeled primer aiming at the new coronavirus open reading frame 1ab is as follows:
a forward primer: 5 '-Biotin-CCCTGTGGGTTTTACACTTAA-3', as shown in SEQ ID NO.1,
reverse primer: 5 '-TAMARA-ACGATTGTGCATCAGCTGA-3' as shown in SEQ ID NO. 2;
the sequence of the specific marker primer aiming at the novel coronavirus encoding nucleocapsid protein N gene is as follows:
a forward primer: 5 '-Biotin-GGGGAACTTCTCCTGCTAGAAT-3', as shown in SEQ ID NO.3,
reverse primer: 5 '-FAM-CAGACATTTTGCTCTCAAGCTG-3' as shown in SEQ ID NO. 4;
the sequence of the specific marker primer aiming at the reference gene RNase P is as follows:
a forward primer: 5 '-Biotin-AGATTTGGACCTGCGAGCG-3', as shown in SEQ ID NO.5,
reverse primer: 5 '-DIG-GAGCGGCTGTCTCCACAAGT-3' as shown in SEQ ID NO. 6.
Preferably, the amplification reaction solution comprises reverse transcriptase, Taq DNA polymerase, 4 dNTPs and ions required by the PCR amplification reaction solution.
Preferably, the positive control is a pseudovirus mixture containing a COVID-19 new coronavirus target gene sequence and an internal reference target sequence.
Preferably, the negative control is Rnase Free ddH2O。
Preferably, the concentration of the specific marker primers for the new coronavirus open reading frame 1ab and the gene encoding the nucleocapsid protein N is 50-400nM each.
Preferably, the concentration of the specific marker primers for the new coronavirus open reading frame 1ab and the gene encoding the nucleocapsid protein N are both 200 nM.
Preferably, the concentration of the specific marker primer for reference gene RNase P is 50-400 nM.
Preferably, the concentration of the specific marker primer for reference gene RNase P is 200 nM.
The invention has the beneficial effects that:
(1) fills up the clinical blank: the novel coronavirus nucleic acid PCR-colloidal gold immunochromatographic assay detection kit can quickly, accurately and sensitively detect the new coronavirus, and fills the blank field of the current clinical detection of the colloidal gold of the new coronavirus nucleic acid;
(2) the clinical diagnosis efficiency is accelerated: the novel coronavirus nucleic acid PCR-colloidal gold immunochromatographic assay kit disclosed by the invention has the advantages of good experimental result repeatability, high precision and short detection time period, can quickly complete detection within 70 minutes, and greatly saves the detection time;
(3) quality control: the quality control strip is designed for the colloidal gold chromatography test paper, so that the quality of the colloidal gold chromatography process can be monitored, and whether false positive occurs or not and manual operation errors can be monitored; and the reference gene is also arranged to carry out whole-process tracking quality control on sample collection, nucleic acid extraction and PCR amplification, and the whole process is supervised;
(4) simple operation, be applicable to basic level hospital: the kit can be used for detection on the computer only by mixing the new coronavirus nucleic acid detection solution with the template by an operator, the detection instrument only depends on a common PCR instrument, the nucleic acid colloidal gold immunochromatography test result is visual, the requirements on operators and instruments are extremely low, and the kit is suitable for high-level, medium-level and low-level hospitals and has high clinical popularization.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments that can be derived by one of ordinary skill in the art from the embodiments given herein are intended to be within the scope of the present invention.
Example 1
A novel coronavirus nucleic acid PCR-colloidal gold immunochromatography detection kit comprises: amplification reaction solution (namely amplification reaction MIX purchased from Nanjing Novozan Biotechnology Co., Ltd. (product number: Q222-CN), the reaction system comprises reverse transcriptase, Taq DNA polymerase, 4 dNTPs and ions required by various PCR amplification reaction solutions), CoV detection solution, positive control (mixed solution of virus target gene plasmid and internal reference plasmid), negative control (RNase Free ddH2O) and chromatography strip (coated colloidal gold particles and antibodies); the CoV detection solution comprises specific marker primers aiming at the open reading frame 1ab of the new coronavirus and the gene encoding the nucleocapsid protein N, specific marker primers of the internal reference gene RNase P and RNase Free ddH2O。
The primer sequences in this example are as follows:
new coronavirus amplification primers:
target one (open reading frame 1ab, sequence shown in SEQ ID No. 7):
forward primer (1ab F): 5 '-Biotin-CCCTGTGGGTTTTACACTTAA-3', as shown in SEQ ID NO.1,
reverse primer (1ab R): 5 '-TAMARA-ACGATTGTGCATCAGCTGA-3' as shown in SEQ ID NO. 2;
target II (encoding nucleocapsid protein N gene, the sequence of which is shown in SEQ ID NO. 8):
forward primer (N F): 5 '-Biotin-GGGGAACTTCTCCTGCTAGAAT-3', as shown in SEQ ID NO.3,
reverse primer (N R): 5 '-FAM-CAGACATTTTGCTCTCAAGCTG-3' as shown in SEQ ID NO. 4;
the 5' ends of the forward primers are all marked by biotin; the 5 'end of the reverse primer of the target one open reading frame 1ab is labeled with TAMARA, and the 5' end of the reverse primer of the target two N is labeled with FAM. The primer concentration is 50-400nM, preferably 200 nM.
An internal reference primer (the sequence of the internal reference gene RNase P is shown as SEQ ID NO. 9):
RNase P-F: 5 '-Biotin-AGATTTGGACCTGCGAGCG-3', as shown in SEQ ID NO.5,
RNase P-R: 5 '-DIG-GAGCGGCTGTCTCCACAAGT-3' as shown in SEQ ID NO. 6;
marking biotin at the 5' end of the internal reference forward primer; the 5' end of the reverse primer can be marked by FITC or DIG, preferably DIG, the primer concentration is 50-400nM, preferably 200nM, and the above components are mixed according to a certain proportion to form the CoV detection solution.
In this example, the new coronavirus open reading frame 1ab, the gene sequence encoding the nucleocapsid protein N and the gene sequence of the internal reference gene RNase P were selected and compared to design a pair of specific primers. Ensures that the three pairs of primers can specifically amplify a new coronavirus open reading frame 1ab, a gene encoding nucleocapsid protein N and an internal reference gene, while other viruses or germs (such as legionella pneumophila, Klebsiella pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae, adenovirus type 3, Mycoplasma pneumoniae, Chlamydia pneumoniae, parainfluenza type 2, respiratory syncytial virus type A, Bordetella pertussis, coronavirus OC43, coronavirus NL63, coronavirus HKU-1, coronavirus 229E, avian influenza virus H7N9, avian influenza virus H5N1, influenza B virus (Victoria line), influenza A H1N1(2009) influenza virus, influenza A H3N2 virus, EB virus, MERS pseudovirus (ORF1ab + N + part of RdRp gene) and negative mock swab samples) can not be amplified.
The components of the kit are shown in table 1, and the kit is stored at-20 ℃.
TABLE 1 Components of the kit
The detection method adopting the kit of the embodiment comprises the following steps: the new coronavirus RNA is taken as a template, an amplification reaction system is prepared by a one-step method (reverse transcription and PCR amplification are completed in the same reaction system) for PCR amplification, and an internal reference gene RNase P is used for carrying out whole-process quality control on sample collection, nucleic acid extraction and PCR amplification.
The amplification reaction system comprises: sample template, amplification reaction solution and CoV detection solution.
The amplification procedure is as follows:
10min at 50 ℃ (1 cycle);
95 ℃ for 3min (1 cycle);
95 ℃ 15sec, 55 ℃ 30sec, 60 ℃ 30sec (35 cycles);
5min at 60 ℃ (1 cycle);
infinity at 4 ℃ (1 cycle);
and (3) performing amplification by using a conventional PCR instrument.
Further, after the PCR reaction is completed, the following operations are sequentially performed:
a. taking a substance to be detected (PCR amplification product) for detection, and diluting the amplification product by 10 times by using a sample diluent for later use, wherein the dilution times can be adjusted according to the concentration of a target substance;
b. taking out the chromatographic test strip and placing the chromatographic test strip on a horizontal desktop (for use as soon as possible), and paying attention not to touch an NC membrane;
c. and (3) sucking 80 mu L of sample to be detected by a pipette or a dropper, slowly and dropwise adding the sample to be detected on the sample adding hole of the test strip, and judging and reading the result after 2-10 minutes.
The results are analyzed as shown in table 2:
TABLE 2 analysis of the results
Example 2
Performance verification of the kit described in example 1
(1) Sample processing
Selecting a purchased national reference product of a novel coronavirus nucleic acid detection reagent to perform kit performance verification, taking a sample to be detected to a 1.5mL centrifuge tube, using a nucleic acid extraction or purification reagent produced by Tiangen Biochemical technology (Beijing) Co., Ltd, and performing RNA extraction operation strictly according to a specification. The extracted RNA samples should be tested immediately or stored below-70 ℃ and repeated freezing and thawing is avoided.
(2) PCR amplification
PCR amplification reaction solutions (20. mu.L per reaction) were prepared as shown in Table 3.
TABLE 3 PCR amplification reaction solution
| Components | 1 reaction volume |
| CoV detection liquid | 7.5μL |
| Amplification reaction solution | 12.5μL |
| Total volume | 20μL |
The prepared PCR amplification reaction solution was dispensed into each reaction well of 20. mu.L. Adding 5 mul each of the extracted sample RNA, positive reference substance RNA and negative reference substance template into corresponding reaction holes, and performing PCR amplification on the reaction holes.
The amplification procedure was:
10min at 50 ℃ (1 cycle);
95 ℃ for 3min (1 cycle);
95 ℃ 15sec, 55 ℃ 30sec, 60 ℃ 30sec (35 cycles);
5min at 60 ℃ (1 cycle);
infinity at 4 ℃ (1 cycle);
and (3) performing amplification by using a conventional PCR instrument.
(3) Chromatography assay
After the PCR reaction is finished, the following operations are carried out in sequence:
a. taking a substance to be detected (PCR amplification product) for detection, and diluting the amplification product by 10 times by using a sample diluent for later use, wherein the dilution times can be adjusted according to the concentration of a target substance;
b. taking out the chromatographic test strip and placing the chromatographic test strip on a horizontal desktop (for use as soon as possible), and paying attention not to touch an NC membrane;
c. and (3) sucking 80 mu L of sample to be detected by a pipette or a dropper, slowly and dropwise adding the sample to be detected on the sample adding hole of the test strip, and judging and reading the result after 2-10 minutes.
(4) Performance of the kit
As shown in table 4, the results of the detection of the positive samples of 7 national references were positive, and the positive compliance rate was 100%.
TABLE 4 Positive references of Xinguan nationality
| Numbering | Pathogens | The result of the detection |
| P1 | Virus cultures | Positive for |
| P2 | Throat swab | Positive for |
| P3 | Virus cultures | Positive for |
| P4 | Throat swab | Positive for |
| P5 | Throat swab | Positive for |
| P6 | Throat swab | Positive for |
| P7 | Plasmid (N full-Length Gene) | Positive for |
As shown in table 5, the national reference samples were tested for 22 pathogenic bacteria or viruses, including legionella pneumophila, klebsiella pneumoniae, streptococcus pneumoniae, haemophilus influenzae, adenovirus type 3, mycoplasma pneumoniae, chlamydia pneumoniae, parainfluenza type 2, respiratory syncytial virus type a, bordetella pertussis, coronavirus OC43, coronavirus NL63, coronavirus HKU-1, coronavirus 229E, avian influenza virus H7N9, avian influenza virus H5N1, influenza b virus (Victoria), influenza a H1N1(2009) influenza virus, influenza a H3N2, EB virus, MERS pseudovirus (ORF1ab + N + part of RdRp gene), and negative mock swab, and the test results were negative, and the coincidence rate was 100%.
TABLE 5 negative reference of New crown nations
Precision: after the standard product R is diluted by 1:20 (19 parts of water +1 part of sample) by using RNA/DNase-free deionized water, the detection is carried out after nucleic acid extraction according to the requirements of kit instructions, and the detection is repeated for 10 times. The results of 10 detections are positive, and the coefficient of variation (CV,%) of the Ct value is not higher than 5%.
Sensitivity: standard substance S (stock solution concentration is 3X 10)5copies/mL) was diluted 1:3 fold (2 parts water +1 part sample) with RNA/dnase-free deionized water, and 1:9, 1:27, 1:81, 1:243, 1:729, 1:2187, 1:6561, 1:19683, 1:59049, and 1:177147 were labeled S1 to S10, respectively, and nucleic acid extraction was performed according to the kit instructions and then detection was performed. The detection results are positive from S1 to S3, and no detection is performed from S4 to S10 (shown in Table 6), thus meeting the requirement of the lowest detection limit.
TABLE 6 sensitivity of New crown national reference
| S | Results | Dilution ratio |
| 1 | Positive for | 1:9 |
| 2 | Positive for | 1:27 |
| 3 | Positive for | 1:81 |
| 4 | Not detected out | 1:243 |
| 5 | Not detected out | 1:729 |
| 6 | Not detected out | 1:2187 |
| 7 | Not detected out | 1:6561 |
| 8 | Not detected out | 1:19683 |
| 9 | Not detected out | 1:59049 |
| 10 | Not detected out | 1:177147 |
In summary, with the aid of the above technical solutions, the novel coronavirus nucleic acid PCR-colloidal gold immunochromatography detection kit provided by the invention is used for extracting nucleic acid from a collected cell sample, amplifying a specific fragment under the action of reverse transcriptase and Taq DNA polymerase, and detecting new coronavirus nucleic acid by colloidal gold immunochromatography. In addition, the kit adopts the reference gene RNaseP to carry out tracking quality control on the collection, nucleic acid extraction and PCR amplification processes of the sample, and ensures that all the experimental processes are in a controllable range. The kit does not depend on an expensive fluorescent quantitative PCR instrument for detecting the new coronavirus nucleic acid amplification product, and the whole operation process is simple, quick, stable and controllable. In addition, the requirement on the quality of the operators is low, and the system can be rapidly and widely popularized and used in low-level, medium-level and high-level hospitals.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
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Claims (8)
1. A novel coronavirus nucleic acid PCR-colloidal gold immunochromatography detection kit is characterized by comprising: amplification reaction solution, CoV detection solution, positive control substance and negative control substance;
the CoVThe detection solution comprises specific marker primers aiming at the open reading frame 1ab of the new coronavirus and the gene encoding the nucleocapsid protein N, specific marker primers of an internal reference gene RNase P and RNase Free ddH2O；
The sequence of the specific labeled primer aiming at the new coronavirus open reading frame 1ab is as follows:
a forward primer: 5 '-Biotin-CCCTGTGGGTTTTACACTTAA-3', as shown in SEQ ID NO.1,
reverse primer: 5 '-TAMARA-ACGATTGTGCATCAGCTGA-3' as shown in SEQ ID NO. 2;
the sequence of the specific marker primer aiming at the novel coronavirus encoding nucleocapsid protein N gene is as follows:
a forward primer: 5 '-Biotin-GGGGAACTTCTCCTGCTAGAAT-3', as shown in SEQ ID NO.3,
reverse primer: 5 '-FAM-CAGACATTTTGCTCTCAAGCTG-3' as shown in SEQ ID NO. 4;
the sequence of the specific marker primer aiming at the reference gene RNase P is as follows:
a forward primer: 5 '-Biotin-AGATTTGGACCTGCGAGCG-3', as shown in SEQ ID NO.5,
reverse primer: 5 '-DIG-GAGCGGCTGTCTCCACAAGT-3' as shown in SEQ ID NO. 6.
2. The PCR-colloidal gold immunochromatographic assay kit for detecting a nucleic acid of a novel coronavirus according to claim 1, wherein the amplification reaction solution comprises a reverse transcriptase,TaqDNA polymerase, 4 kinds of dNTPs and ions needed by PCR amplification reaction liquid.
3. The PCR-colloidal gold immunochromatographic assay kit for detecting a novel coronavirus nucleic acid according to claim 1, wherein the positive control is a pseudovirus mixture containing a COVID-19 novel coronavirus target gene sequence and an internal reference target sequence.
4. The kit for detecting coronavirus nucleic acid PCR-colloidal gold immunochromatography according to claim 1, wherein the negative control is Rnase Free ddH2O。
5. The PCR-colloidal gold immunochromatographic assay kit for detecting a novel coronavirus nucleic acid according to claim 1, wherein the concentration of the specific marker primers for the novel coronavirus open reading frame 1ab and the gene encoding nucleocapsid protein N is 50-400 nM.
6. The PCR-colloidal gold immunochromatographic assay kit for detecting a novel coronavirus nucleic acid according to claim 5, wherein the concentration of the specific marker primers for the novel coronavirus open reading frame 1ab and the gene encoding nucleocapsid protein N is 200 nM.
7. The PCR-colloidal gold immunochromatographic assay kit for detecting a novel coronavirus nucleic acid according to claim 1, wherein the concentration of the specific labeled primer for the reference gene RNase P is 50-400 nM.
8. The PCR-ELISA detection kit of claim 7 wherein the concentration of the specific labeled primer for reference gene RNase P is 200 nM.
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