CN111500776A - Novel coronavirus 2019-nCoV fluorescent RPA detection primer, probe, kit and method - Google Patents
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Abstract
The invention discloses a novel coronavirus 2019-nCoV fluorescent RPA detection primer, a probe, a kit and a method. It comprises a novel coronavirus 2019-nCoV specific primer shown as SEQ ID NO.1-2 and a probe shown as SEQ ID NO. 3. The invention adopts a single-tube double-fluorescence channel to simultaneously detect the existence of the novel coronavirus 2019-nCoV ORF1ab gene and the reference gene RNase P, and can detect the existence of the novel coronavirus 2019-nCoV RNA in samples such as alveolar lavage fluid, nasal swab, pharyngeal swab, sputum and the like. The invention has short detection time and is suitable for clinical and bedside virus nucleic acid rapid screening; has extremely high sensitivity and specificity; and adding an internal reference gene into the detection system, and performing quality monitoring on the sample extraction and amplification process according to the detection result of the internal reference gene.
Description
Technical Field
The invention belongs to the field of biotechnology and molecular diagnosis, and particularly relates to a novel primer, a probe, a kit and a method for detecting coronavirus 2019-nCoV fluorescent RPA
Background
The world health organization named 2019 novel coronavirus, namely "2019-nCoV", in a case of viral pneumonia 12 months since 2019. the novel coronavirus 2019-nCoV belongs to the family of coronaviridae, the genus beta coronavirus, is enveloped, has particles in a circular or oval shape, is usually polymorphic, has a diameter of 60-140nm, and is the 7 th coronavirus which is currently known to infect humans, and the remaining 6 coronaviruses are HCoV-229E, HCoV-OC43, HCoV-N L63, HCoV-HKU 1, SARS-CoV and MERS-CoV, wherein the first 4 coronaviruses are common in the human population, have low pathogenicity, and generally cause only slight respiratory symptoms similar to common cold, and the other 2 coronaviruses are SARS coronavirus and MERS coronavirus (respiratory syndrome coronavirus).
At present, the main method for detecting nucleic acid is a PCR method, and the conventional PCR detection needs a special thermal cycler and a complicated experimental procedure and is difficult to meet the detection requirements on the spot or under other conditions outside a laboratory. In recent years, isothermal amplification techniques for nucleic acids have been developed, which do not require an expensive thermal cycling PCR instrument, can rapidly amplify a large amount of target fragments in a short time, and have the advantages of simplicity, rapidness, high sensitivity, and the like. Among them, the RPA technique is more referred to as a nucleic acid detection technique that can replace conventional PCR. RPA technology relies primarily on three enzymes: recombinases that bind single-stranded nucleic acids (oligonucleotide primers), single-stranded DNA binding proteins (SSBs), and strand-displacing DNA polymerases. The mixture of the three enzymes has activity at normal temperature, and the optimal reaction temperature is about 37-42 ℃. RPA technology has been developed based on the principle of recombinase polymerase-mediated amplification, which mimics DNA replication in organisms. It can increase the target gene exponentially in a very short time, and if the fluorescent-labeled probe and the fluorescent signal detector are matched, the real-time monitoring of the template amplification can be realized. The RPA technology greatly reduces the detection time, simplifies the reaction procedure, is very suitable for the field detection of places such as bedside, airport, customs import and export and the like after combining the rapid nucleic acid extraction technology and the portable real-time fluorescence detection equipment, and has wide application prospect.
Disclosure of Invention
The invention aims to provide a primer, a probe, a kit and a method for detecting fluorescent RPA of novel coronavirus 2019-nCoV aiming at the whole genome sequence information of the novel coronavirus 2019-nCoV, so that the requirements of quick, sensitive and accurate detection are met.
The purpose of the invention can be realized by the following technical scheme:
a novel coronavirus 2019-nCoV fluorescent RPA detection primer and a probe comprise a novel coronavirus 2019-nCoV specific primer shown as SEQ ID NO.1-2 and a probe shown as SEQ ID NO. 3.
The fluorescent RPA detection primer and the probe are applied to preparation of a novel coronavirus 2019-nCoV fluorescent RPA detection kit.
A novel coronavirus 2019-nCoV fluorescent RPA detection kit comprises the fluorescent RPA detection primer and a probe.
As a preferred technical scheme, the kit also comprises specific primers of an internal reference gene RNase P shown in SEQ ID NO.4-5 and specific probes of an internal reference gene RNase P shown in SEQ ID NO. 6.
Further preferably, the kit further comprises:
positive control: plasmid containing new type coronavirus 2019-nCoV ORF1ab gene sequence;
negative control: RNase Free H2O。
The detection method of the novel coronavirus 2019-nCoV fluorescent RPA detection kit comprises the steps of taking RNA of a sample to be detected as a template, preparing an amplification reaction system, carrying out fluorescent RPA amplification to obtain an amplification curve, analyzing the amplification curve, and judging; the amplification reaction system comprises the fluorescent RPA detection primer and the probe of the novel coronavirus 2019-nCoV.
As a preferred technical scheme, the amplification reaction system comprises: an RNA template of a sample to be detected, the novel coronavirus 2019-nCoV fluorescent RPA detection primer and probe, RPA Buffer (reaction Buffer), Enzyme Mix (Enzyme mixed solution) and an activator. Further preferred is: the amplification reaction system also comprises an internal reference gene RNase P specific primer shown as SEQ ID NO.4-5 and an internal reference gene RNase P specific probe shown as SEQ ID NO. 6.
The procedure for the fluorescent RPA amplification was: fluorescence was collected once at intervals of 30s at 40 ℃ for 20min for 40 cycles.
The principle of analyzing and judging the amplification curve is as follows:
when the time value in the FAM fluorescence channel is less than or equal to 19min, judging that the sample is positive to the novel coronavirus 2019-nCoV;
when the time value is less than or equal to 20min after 19min in the FAM fluorescence channel, repeating the experiment once, if the time value is still within the range or less than or equal to 19min, judging the sample to be the new coronavirus 2019-nCoV positive, otherwise, judging the sample to be the new coronavirus 2019-nCoV negative;
when the FAM fluorescence channel has no amplification curve and the HEX channel time value is less than or equal to 20min, judging that the sample is negative to the novel coronavirus 2019-nCoV;
when the FAM fluorescence channel has no amplification curve and the HEX channel also has no amplification curve, the experiment is judged to be abnormal, and the RNA of the sample needs to be extracted again and amplified again.
In summary, the advantages and positive effects of the invention are:
1. the invention provides a fluorescent RPA detection primer pair, a probe, a kit and a detection method for a novel coronavirus 2019-nCoV, which can detect the novel coronavirus 2019-nCoV more quickly, accurately and sensitively, and the detection rate is as high as 5copies/m L.
2. According to the invention, a large number of experiments are carried out to screen out the primer pair, the probe, the kit and the detection method, the single-tube double-fluorescence channel is adopted to simultaneously detect the existence of the novel coronavirus 2019-nCoV and the internal reference gene RNase P, and the existence of the novel coronavirus 2019-nCoV RNA in samples such as alveolar lavage fluid, nasal swab, pharyngeal swab, sputum and the like can be detected. When reverse transcription or RPA amplification inhibition exists in alveolar lavage fluid, nasal swab, pharyngeal swab and sputum, the detection result of viral nucleic acid is inaccurate and even false negative easily occurs. The invention adopts high-efficiency reverse transcriptase and polymerase, combines a rapid nucleic acid extraction technology and portable constant-temperature fluorescence detection equipment, simplifies reaction procedures, reduces experiment time, can obtain detection results within 20min, shortens about 2h compared with the existing nucleic acid detection method, meets the requirements of rapid and accurate diagnosis, and is very suitable for field detection at places such as bedside, airports, customs imports and exports, and the like.
Drawings
FIG. 114 example pharyngeal swab sample novel coronavirus 2019-nCoV fluorescent RPA amplification curve;
FIG. 214 is a reference gene RNase P fluorescence RPA amplification curve of throat swab sample;
FIG. 3 fluorescent RPA amplification curves of 2019-nCoV virus from different types of samples from patients diagnosed with the novel coronavirus 2019-nCoV;
FIG. 4 fluorescent RPA amplification curves of RNase P, an internal reference gene, from different types of samples from patients diagnosed with the novel coronavirus 2019-nCoV.
Detailed Description
The present invention will be described in detail with reference to specific examples. From the following description and these examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.
Example 1 development of fluorescent RPA detection kit
1. In the embodiment, a pair of specific primers and a specific fluorescent probe are designed from a novel coronavirus 2019-nCoV specific open reading frame ORF1ab gene sequence, an internal standard gene RNase P is selected to design a pair of specific primers and a specific fluorescent probe, and a fluorescent RPA technology is constructed to detect the novel coronavirus 2019-nCoV, wherein the ORF1ab gene is an important structural gene of the coronavirus, and the primers and the probes designed by the invention are only similar to 100% of the ORF1ab gene of the novel coronavirus 2019-nCoV and are not matched with other coronaviruses including SARS, MERS, 229E, OC43, N L63 and HKU1, so that the primers and the probe sequences can be specifically combined with the ORF1ab gene of the novel coronavirus 2019-nCoV and can be amplified without amplifying other types of coronaviruses.
TABLE 1 primer Probe sequences
The kit also comprises a positive control and a negative control: RNase Free H2O, RPA Buffer, Enzyme Mix, activator.
① RPA Buffer configuration:
20×Core Reaction Mix 1.25μL
2×Reaction Buffer 12.5μL
dNTP 1.25μL
mixing, and storing at-20 deg.C.
② Enzyme Mix configuration:
10×E-Mix 2.5μL
50×exoⅢ 0.5μL
50×RT 0.5μL
mixing, and storing at-20 deg.C.
2. Fluorescent RPA detection method of novel coronavirus 2019-nCoV
① extraction of viral nucleic acid
And (3) extracting the nucleic acid of 14 pharyngeal swab samples of suspected respiratory virus infected patients by using a commercial virus RNA extraction kit.
② fluorescent RPA amplification
Taking 4 mu L RNA as a template, simultaneously adding a No. 19 mu L1-8 RPA reaction solution reagent (the preparation system of the fluorescent RPA reaction solution is shown in table 2) into an eight-connected tube, adding 2 mu L activator into an eight-connected tube cover, covering the tube cover, performing instantaneous centrifugation, and performing fluorescent RPA amplification on a machine.
TABLE 2 preparation system of real-time fluorescent quantitative PCR reaction solution
| Serial number | Name of reagent | Volume of |
|
| 1 | RPA Buffer | 15μL | |
| 2 | Enzyme Mix | 3.5μL | |
| 3 | ORF1ab F | 0.1 |
|
| 4 | ORF1ab R | 0.1 |
|
| 5 | ORF1ab P | 0.05μL | |
| 6 | RNaseP F | 0.1μL | |
| 7 | RNaseP R | 0.1μL | |
| 8 | RNaseP P | 0.05μL | |
| 9 | | 4μL | |
| 10 | Activating agent | 2μL |
③ fluorescent RPA reaction program
The fluorescent RPA reaction program was: fluorescence was collected once at intervals of 30s at 40 ℃ for 20min for 40 cycles. The present invention uses a 1610-Q fluorescence detector for detection.
④ obtaining the result of the fluorescent RPA detection, analyzing the amplification curve and making a judgment, the judgment rule is:
when the time value in the FAM fluorescence channel is less than or equal to 19min, judging that the sample is positive to the novel coronavirus 2019-nCoV;
when the time value of 19< in the FAM fluorescence channel is less than or equal to 20min, repeating the experiment once, if the Ct is still within the range or less than or equal to 19min, judging that the sample is the new coronavirus 2019-nCoV positive, otherwise, judging that the sample is the new coronavirus 2019-nCoV negative;
when the FAM fluorescence channel has no amplification curve and the time value in the HEX channel is less than or equal to 20min, judging that the sample is negative to the novel coronavirus 2019-nCoV;
when the FAM fluorescence channel has no amplification curve and the HEX channel also has no amplification curve, the experiment is judged to be abnormal, and the RNA of the sample needs to be extracted again and amplified again.
3. Results of the experiment
Detecting 14 parts of throat swab samples of suspected respiratory virus infected patients by using the constructed method, and detecting 1 positive example of the novel coronavirus 2019-nCoV; FIG. 1 shows the virus fluorescence RPA amplification curves of the 1 new coronavirus 2019-nCoV positive specimen and the 13 negative specimens, and meanwhile, the amplification curve of the internal reference gene RNase P in the 14 specimens is normal, as shown in FIG. 2, which shows that the extraction and amplification process of the experiment is normal, and the positive and negative results are accurate.
Example 2 Performance testing of fluorescent RPA detection kit for the novel coronavirus 2019-nCoV
1. Accuracy verification
The gold standard of virus nucleic acid detection is genome sequencing, and the detection result of the secondary kit is compared with the virus genome sequencing result to analyze the accuracy of the detection result. In this example, 4 samples determined to be the novel coronavirus 2019-nCoV by genome sequencing were selected, and the results of detection by the kit provided by the present invention are shown in Table 3 below. Therefore, 4 positive samples are detected, and the accuracy of the novel coronavirus 2019-nCoV fluorescent RPA detection kit provided by the invention is 100%.
TABLE 3 analysis of the accuracy of the invention
| Class of viruses | Number of examples | Positive in sequencing | Fluorescent RPA method | Rate of accuracy |
| Novel coronavirus 2019- |
4 | 4 | 4 | 100％ |
2. Specificity verification
The specificity of the kit was assessed by detecting other pathogens, and 14 positive specimens of respiratory tract and other common pathogens or plasmid-mimic positive specimens were selected in this example, with the results shown in table 4 below. Through detection, the kit has no amplification on 14 respiratory tract and other common pathogen positive specimens, and shows that the specificity of the novel coronavirus 2019-nCoV fluorescent RPA detection kit provided by the invention is 100%.
TABLE 4 specificity assay of the invention
3. Sensitivity verification
The sensitivity, namely the lowest detection limit, means that after a positive sample is subjected to gradient dilution, the detection rate of the sample with the lowest dilution gradient is more than or equal to 95%, the detection frequency of the sample for sensitivity evaluation at each concentration level to be evaluated is not less than 20, and a positive amplification curve is qualified after at least 19 times of detection, wherein after a positive standard product of the novel coronavirus 2019-nCoV is subjected to gradient dilution according to a certain copy number, each dilution degree is averagely divided into 20 samples, the detection is carried out by using the method disclosed by the invention, the copy number of the positive samples with the number of 19 times or more is the lowest detection limit, the result is shown in a table 5, and the detection rate is 95% when the concentration is 5copies/m L, and is less than 95% when the concentration is lower, so that the sensitivity of the fluorescent RPA detection kit for the novel coronavirus 2019-nCoV provided by the invention is 5copies/m L.
TABLE 5 sensitivity assay of the invention
4. Detection of precision
The precision refers to that the same positive sample is adopted for multiple detections, the consistency of interpretation results is judged to be good precision by the Coefficient of Variation (CV) being less than 3. in the embodiment, 4 positive samples are detected for 3 times in each concentration gradient, and the results are shown in the following table 6. the results are verified to be 5-1 × 108In copy/m L range, batch CV of this invention<3% and inter-batch CV<3%, and the precision is good.
TABLE 6 analysis of precision of the invention
EXAMPLE 3 clinical trials
The results of the detection of alveolar lavage fluid, nasal swab and pharyngeal swab sputum from the novel coronavirus 2019-nCoV positive confirmed patient by the method are shown in FIG. 3, and the alveolar lavage fluid, nasal swab, pharyngeal swab and sputum from the novel coronavirus 2019-nCoV positive confirmed patient have amplification curves. Meanwhile, the amplification curves of the internal reference gene RNase P of the specimens are normal, as shown in FIG. 4, which shows that the nucleic acid extraction and amplification processes of the experiment are normal, and the positive and negative results are accurate. Therefore, the method is proved to be suitable for detecting alveolar lavage fluid, nasal swab, pharyngeal swab and sputum of a suspected patient infected by the novel coronavirus 2019-nCoV.
The invention is not to be considered as limited to the particular embodiments shown, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. A novel primer and a probe for detecting coronavirus 2019-nCoV fluorescent RPA are characterized by comprising a novel primer for detecting coronavirus 2019-nCoV shown as SEQ ID NO.1-2 and a probe shown as SEQ ID NO. 3.
2. The use of the fluorescent RPA detection primers and probes of claim 1 in the preparation of novel coronavirus 2019-nCoV fluorescent RPA detection kits.
3. A novel coronavirus 2019-nCoV fluorescent RPA detection kit, which is characterized by comprising the fluorescent RPA detection primer and probe in claim 1.
4. The novel coronavirus 2019-nCoV fluorescent RPA detection kit as claimed in claim 3, wherein the kit further comprises an internal reference gene RNase P specific primer shown in SEQ ID NO.4-5 and an internal reference gene RNase P specific probe shown in SEQ ID NO. 6.
5. The novel coronavirus 2019-nCoV fluorescent RPA detection kit according to claim 3 or 4, which is characterized by further comprising:
positive control: plasmid containing new type coronavirus 2019-nCoV ORF1ab gene sequence;
negative control: RNase Free H2O。
6. The detection method of the novel coronavirus 2019-nCoV fluorescent RPA detection kit is characterized in that a sample RNA to be detected is taken as a template, an amplification reaction system is prepared for carrying out fluorescent RPA amplification to obtain an amplification curve, the amplification curve is analyzed, and judgment is made; the amplification reaction system comprises the fluorescent RPA detection primer and the probe of the novel coronavirus 2019-nCoV as claimed in claim 1.
7. The method for detecting the novel coronavirus 2019-nCoV fluorescent RPA detection kit according to claim 6, which is characterized in that: the amplification reaction system comprises: an RNA template of a sample to be detected, the novel coronavirus 2019-nCoV fluorescent RPA detection primer and probe as claimed in claim 1, RPA Buffer, Enzyme Mix and an activator.
8. The detection method of the novel coronavirus 2019-nCoV fluorescent RPA detection kit according to claim 7, which is characterized in that: the amplification reaction system also comprises an internal reference gene RNase P specific primer shown as SEQ ID NO.4-5 and an internal reference gene RNase P specific probe shown as SEQ ID NO. 6.
9. The method for detecting the novel coronavirus 2019-nCoV fluorescent RPA detection kit according to claim 6, which is characterized in that: the procedure for the fluorescent RPA amplification was: fluorescence was collected once at intervals of 30s at 40 ℃ for 20min for 40 cycles.
10. The method for detecting the novel coronavirus 2019-nCoV fluorescent RPA detection kit according to claim 6, wherein the principle of analyzing and judging the amplification curve is as follows:
when the time value in the FAM fluorescence channel is less than or equal to 19min, judging that the sample is positive to the novel coronavirus 2019-nCoV;
when the time value is less than or equal to 20min after 19min in the FAM fluorescence channel, repeating the experiment once, if the time value is still within the range or less than or equal to 19min, judging the sample to be the new coronavirus 2019-nCoV positive, otherwise, judging the sample to be the new coronavirus 2019-nCoV negative;
when the FAM fluorescence channel has no amplification curve and the HEX channel time value is less than or equal to 20min, judging that the sample is negative to the novel coronavirus 2019-nCoV;
when the FAM fluorescence channel has no amplification curve and the HEX channel also has no amplification curve, the experiment is judged to be abnormal, and the RNA of the sample needs to be extracted again and amplified again.
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Cited By (10)
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| CN112266980A (en) * | 2020-11-03 | 2021-01-26 | 郑州大学 | Novel coronavirus 2019-nCoV real-time fluorescent PCR detection primer, probe, kit and method |
| CN112458210A (en) * | 2020-12-09 | 2021-03-09 | 上海伯杰医疗科技有限公司 | Gene conserved sequence, primer probe combination, kit and application for detecting new coronavirus |
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| CN112877470A (en) * | 2020-12-11 | 2021-06-01 | 肇庆大华农生物药品有限公司 | New coronavirus RPA reaction system and application thereof |
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