CN117265180A - RT-LAMP primer and probe combination for detecting novel coronavirus, kit and application of kit - Google Patents
RT-LAMP primer and probe combination for detecting novel coronavirus, kit and application of kit Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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Abstract
The invention provides an RT-LAMP primer and probe combination for detecting a novel coronavirus, which belongs to the technical field of virus detection and has the characteristics of strong specificity and high accuracy when detecting the novel coronavirus. The kit for detecting the novel coronavirus based on the RT-LAMP technology has the advantages of low cost, easy acquisition of used reagents and low cost. Compared with the industrial detection gold standard RT-qPCR detection method, the method can achieve similar detection sensitivity, realize rapid, sensitive, accurate and simple detection, and improve detection efficiency.
Description
Technical Field
The invention belongs to the technical field of virus detection, and particularly relates to an RT-LAMP primer and probe combination for detecting a novel coronavirus, a kit and application thereof.
Background
The new coronavirus is also called a novel coronavirus, which has been named as "SARS-CoV-2" by the International Commission on classification of viruses, and SARS-CoV-2 is a single-stranded positive strand RNA virus with high variability in genome. Symptoms of patients infected with the new coronavirus are typically fever, weakness, dry cough, gradual onset of dyspnea, severe cases manifested as acute respiratory distress syndrome, septic shock, uncorrectable metabolic acidosis and coagulation dysfunction. In addition, the novel coronaviruses are extremely infectious and infectious in the latent period, so that the most effective methods for controlling the novel coronavirus infection are early discovery, early isolation, early diagnosis and early treatment.
The detection of novel coronavirus nucleic acid positives using reverse transcription real-time fluorescent quantitation (RT-qPCR) remains the established gold standard, as prescribed by the latest guidelines. However, when using RT-qPCR detection, the supply shortages of sample collection reagents, RNA extraction reagents, RT-qPCR reagents, automated laboratory fluid handling machines and thermocyclers are common, and the instrumentation required for detection is expensive and a skilled technician, the detection time is long, and the test cost is high. Therefore, it is important to develop a rapid, accurate, simple and convenient detection kit for SARS-CoV-2.
The loop-mediated isothermal amplification (LAMP) detection technology is characterized in that 4 specific primers are designed for 6 regions of a target gene, and nucleic acid amplification is carried out under the action of a DNA polymerase (BstDNApolymerase) at a constant temperature, so that the method has the characteristics of simplicity in operation and strong specificity. The LAMP detection technology has the advantages of no need of temperature change, high amplification speed, low instrument requirement, simplicity and easiness in operation, and rapid diagnosis by general personnel without professional skills. However, current LAMP detection techniques still suffer from the drawbacks of requiring RNA extraction or risk of environmental cross-contamination, high cost, and being able to detect only in limited sample types and patient populations, thus limiting their flexible use in scale monitoring tests.
Disclosure of Invention
In view of the above, the invention aims to provide an RT-LAMP primer and probe combination for detecting a novel coronavirus, a kit and application thereof, and the kit has the characteristics of rapidness, simplicity, convenience, accuracy and low cost.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an RT-LAMP primer and probe combination for detecting a novel coronavirus, which comprises one or more of a primer and a probe for detecting an N gene of the novel coronavirus, a primer and a probe for detecting an E gene of the novel coronavirus and a primer and a probe for detecting an ORF1A gene of the novel coronavirus;
the primer sequences of the primer and the probe of the N gene are shown as SEQ ID No. 1-4, and the probe sequences are shown as SEQ ID No. 5;
the primer sequences of the primer and the probe of the E gene are shown in SEQ ID No. 6-9, and the probe sequences are shown in SEQ ID No. 10;
the primer sequences of the primer and the probe of the ORF1A gene are shown as SEQ ID No. 11-14, and the probe sequence is shown as SEQ ID No. 15.
The invention provides application of the primer and probe combination in preparing a kit for detecting a novel coronavirus.
The invention provides a kit for detecting a novel coronavirus based on an RT-LAMP technology, which comprises the primer and probe combination.
Preferably, the kit further comprises an RNA extraction reagent.
Preferably, the RNA extraction reagent comprises a lysate comprising TCEP-HCl 0.5-1 mg/. Mu.L, 0.4-0.6M EDTA and guanidine hydrochloride 150-250 mM, pH < 9.
Preferably, the kit further comprises a positive control and a negative control, wherein the positive control is a novel coronavirus, and the negative control is DEPC water.
Preferably, the sample of the novel coronavirus used in the assay of the kit is derived from saliva, a nasal or nasopharyngeal swab.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides an RT-LAMP primer and probe combination for detecting a novel coronavirus, which has the characteristics of strong specificity and high accuracy when detecting the novel coronavirus. The kit for detecting the novel coronavirus based on the RT-LAMP technology has the advantages of low cost, easy acquisition of used reagents and low cost. Compared with an industrial detection gold standard RT-qPCR detection method, the method can achieve similar detection sensitivity.
Drawings
FIG. 1 is a measurement of the detection limit of RT-LAMP detection method in nasopharyngeal swab samples;
FIG. 2 is a measurement of the detection limit of RT-LAMP detection method in saliva samples;
FIG. 3 is an assay of the detection limits of the RT-qPCR detection method in nasopharyngeal swab samples;
FIG. 4 is a measurement of the limit of detection of RT-qPCR detection methods in saliva samples;
wherein the abscissa of FIGS. 1 to 4 shows the number of pseudoviruses replicated per reaction.
Detailed Description
The invention provides an RT-LAMP primer and probe combination for detecting a novel coronavirus, which comprises one or more of a primer and a probe for detecting an N gene of the novel coronavirus, a primer and a probe for detecting an E gene of the novel coronavirus and a primer and a probe for detecting an ORF1A gene of the novel coronavirus;
the primer sequences of the primer and the probe of the N gene are shown as SEQ ID No. 1-4, and the probe sequences are shown as SEQ ID No. 5;
the primer sequences of the primer and the probe of the E gene are shown in SEQ ID No. 6-9, and the probe sequences are shown in SEQ ID No. 10;
the primer sequences of the primer and the probe of the ORF1A gene are shown as SEQ ID No. 11-14, and the probe sequence is shown as SEQ ID No. 15.
Further preferably, the RT-LAMP primer and probe combination comprises three of a primer and probe for detecting N gene of a novel coronavirus, a primer and probe for detecting E gene of the novel coronavirus and a primer and probe for detecting ORF1A gene of the novel coronavirus, and the three primer and probe combinations are used in RT-LAMP detection, so that the detection can aim at different areas of SARS-CoV-2 virus genome, and the possibility that virus mutation prevents amplification is reduced.
In the invention, the primer and probe combination for detecting the novel coronavirus takes the novel coronavirus specific N gene, E gene and ORF1A gene as target sequences to design RT-LAMP primers and probes. The nucleotide sequence of the N gene is as the accession number of NCBI: 43740575, the nucleotide sequence of the E gene is shown as the sequence of NCBI accession No. 43740570, and the nucleotide sequence of the ORF1A gene is shown as the sequence of NCBI accession No. 43740578. The primer and probe combination provided by the invention has the characteristics of strong specificity, high sensitivity and high accuracy when detecting the novel coronavirus.
The invention provides application of the primer and probe combination in preparing a kit for detecting a novel coronavirus.
The invention provides a kit for detecting a novel coronavirus based on an RT-LAMP technology, which comprises the primer and probe combination.
In the present invention, the kit further preferably includes an RNA extraction reagent. The RNA extraction reagent preferably comprises a lysate and an RNA purification extraction reagent.
In the present invention, the lysate preferably comprises TCEP EDTA/sodium hydroxide lysis buffer and guanidine hydrochloride, and the lysate preferably comprises TCEP-HCl 0.5-1 mg/. Mu.L, 0.4-0.6M EDTA and guanidine hydrochloride 150-250 mM, pH < 9. Further preferably, the lysate comprises TCEP-HCl 0.63 mg/. Mu.L, 0.5M EDTA and 200mM guanidine hydrochloride, pH 8. The preparation method of the TCEP EDTA/sodium hydroxide lysis buffer solution is preferably to dissolve TCEP-HCl in enzyme-removed water, then add EDTA, and adjust the pH value to be less than 9 by sodium hydroxide. The lysate of the invention has high lysis efficiency and can thoroughly inactivate RNases.
In the present invention, the kit further preferably comprises a positive control and a negative control, wherein the positive control is preferably a new coronavirus, the negative control is preferably DEPC water, the source of the new coronavirus is not particularly limited, and the new coronavirus can be prepared or purchased by a preparation method in the art, such as the new coronavirus can be purchased from NATtrol company TM SARS-associated coronavirus 2.
In the present invention, the sample of the novel coronavirus used in the detection of the kit is preferably derived from saliva, nasal or nasopharyngeal swab. The nasopharyngeal swab is adopted as the sample in the sample to detect the nasopharyngeal swab, so that the detection result is more accurate. The sample volume used in the RT-LAMP reaction of the invention is preferably 3.75. Mu.L, which is designed to maximize viral input while achieving the best detection results. Compared with the existing gold standard-RT-qPCR detection method for detecting the novel coronavirus, the sample volume used in the RT-qPCR reaction is preferably 5 mu L, and the sample volume used in the RT-LAMP technology is smaller.
In the present invention, the method of using the kit preferably comprises the steps of:
(1) Preparing an RT-LAMP reaction system: the total volume was 25. Mu.L, 2X E1700 Master Mix 12.5. Mu.L, 25X N2 primer Mix 1. Mu.L, 25X E1 primer Mix 1. Mu.L, 50X fluorescent dye (NEB 1700) 0.5. Mu.L and template 10. Mu.L.
(2) Carrying out LAMP reaction on the prepared RT-LAMP reaction system, wherein the reaction procedure is that the reaction is carried out for 45min at 65 ℃, and the reaction is finished after the inactivation is carried out for 6min at 96 ℃;
(3) A determination is made as to whether the new coronavirus is contained based on the data value.
In the invention, the sample is detected, and if the Ct value of the sample is smaller than 30, the sample is judged to be a new coronavirus positive sample.
In the present invention, 5 in nasopharyngeal samples, the limit of detection was 20 viral particles per reaction on Bio-Rad CFX 96 and 23 viral particles per reaction on AxxinT 8. In saliva, the detection limit of Bio-Rad CFX 96 was 93 virus particles per reaction, and the detection limit of Axin T8 was 116 virus particles per reaction.
In the invention, clinical samples are used as detection objects, and the RT-LAMP detection kit and the RT-qPCR detection method provided by the invention are respectively used for detection, so that the results show that the detection results obtained by the two detection methods of 50 clinical samples are completely consistent. The detection kit provided by the invention can replace the existing detection method for detecting the new coronavirus by adopting the RT-qPCR detection method, shortens the detection time, has low cost, and is favorable for efficiently, quickly and accurately realizing the detection of suspicious positive people.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
(1) Oligonucleotide primer and probe design
Primers and probes were designed for the N, E and ORF1A genes of the novel coronavirus SARS-CoV-2, specific primer sequence information is shown in Table 1, and specific probe sequence information is shown in Table 2.
TABLE 1 primer sequence information
TABLE 2 Probe sequence information
(2) Extracting RNA with RNA extraction reagent
Taking 3.75 mu L of nasopharyngeal swab sample, adding 500 mu L of lysate, vortexing for 20 seconds, and purifying RNA by using an RNA purification kit, wherein the lysate is TCEP-HCl 0.63 mg/mu L, 0.5M EDTA and guanidine hydrochloride 200mM, and the pH value is 8.
(3) RT-LAMP amplification reaction of novel coronaviruses
Detection of Nattrol by RT-LAMP technique TM The reaction system of SARS-related coronavirus 2, RT-LAMP was 25. Mu.L, and the reaction system was 2X E1700 MasterMix 12.5. Mu.L, 25X N2 primer Mix 1. Mu.L, 25X E1 primerMix 1. Mu.L, 50X fluorescent dye (NEB 1700) 0.5. Mu.L, and template 10. Mu.L. The reaction procedure of RT-LAMP was 45min at 65℃and 6min at 96℃for inactivation.
The result shows that the primer and probe combination can obtain CT value, which proves that the invention successfully establishes a method for detecting the novel coronavirus based on RT-LAMP technology.
Example 2
Assessment of detection limit of RT-LAMP assay
2.1 The limit of detection of the RT-LAMP assay was assessed by dilution of zeptometric pseudoviruses in pooled negative nasopharyngeal swabs or pooled negative saliva.
Nattrol TM The SARS related coronavirus 2 is respectively diluted by a negative nasopharyngeal swab or negative saliva in a gradient way, and the copy number is sequentially 1-10 copies/. Mu.L. The detection was performed using the RT-LAMP method established in example 1, 20 replicates were performed for each concentration, and the percent replicates for positive amplification at each concentration were calculated. The detection limit is then determined using probe analysis. XLSTAT is Microsoft Excel and the limit of detection is defined as the concentration at which the probability of detection is 95%.
As shown by the results in FIG. 1, in nasopharyngeal swab sample detection, the minimum of 23 copies per reaction of the RT-LAMP assay; as shown by the results in FIG. 2, the minimum of 116 copies per reaction in RT-LAMP assay was observed in saliva sample detection.
2.2 comparison of RT-LAMP detection and RT-qPCR detection methods
RT-qPCR limits were assessed by dilution of Zeptometric pseudoviruses in pooled negative nasopharyngeal swabs or pooled negative saliva, see section 2.1 for specific assessment.
The RT-qPCR detection method comprises the following steps:
the amplification primer and probe information for the novel coronavirus SARS-CoV-2 gene N, E and ORF1A are shown in Table 3.
TABLE 3 amplification primers and Probe information for novel coronavirus genes
| Primer/probe name | (5 '. Fwdarw.3') sequences | Sequence numbering |
| Ngene-F | TGGCTACTACCGAAGAGCT | SEQ ID No.16 |
| Ngene-R | TGCAGCATTGTTAGCAGGAT | SEQ ID No.17 |
| Ngene-Probe | FAM-ACTGAGGGAGCCTTGAATACA-BHQ1 | SEQ ID No.18 |
| Egene-F | TGAGTACGAACTTATGTACTCAT | SEQ ID No.19 |
| Egene-R | TTCAGATTTTTAACACGAGAGT | SEQ ID No.20 |
| Egene-Probe | FAM-GCGCTTCGATTGTGTGCGT-BHQ1 | SEQ ID No.21 |
| ORF1A-F | CGGTGGACAAATTGTCAC | SEQ ID No.22 |
| ORF1A-R | CTTCTCTGGATTTAACACACTT | SEQ ID No.23 |
| ORF1A-Probe | FAM-TTGAATTTAGGTGAAACATTTGTCACG-BHQ1 | SEQ ID No.24 |
The RT-qPCR amplification system contained 1. Mu.L of 2 XRT-pcr Mix, 0.5. Mu.L of 40 XRT Enzyme Mix, 1.5. Mu.L of combined primer/probe Mix, 3. Mu.L of nuclease free water, and 5. Mu.L of purified RNA template for each reaction.
The RT-qPCR amplification procedure was as follows: 15min at 50 ℃, 15min at 95 ℃ and 1 cycle; 94℃15s,55℃45s,40 cycles.
From the results in FIG. 3, it is shown that in nasopharyngeal swab sample detection, RT-qPCR measures a minimum of 20 copies of the new coronavirus per reaction; the results in FIG. 4 show that in saliva sample assays, the minimum response of the RT-qPCR assay is 93 copies per time.
As can be seen from the results of FIGS. 1 to 4, compared with the industrial detection gold standard RT-qPCR detection method, the RT-LAMP detection method can achieve similar detection sensitivity.
Example 3
Assessing accuracy of RT-LAMP assay
69 clinical nasopharyngeal swab samples, 42 nasal swab samples and 35 saliva samples were selected for RT-LAMP and RT-qPCR detection, wherein the method for detecting the novel coronavirus by RT-LAMP is described in example 1, and the method for detecting the novel coronavirus by RT-qPCR is described in example 2.
For nasopharyngeal swabs, when Ct < 30, the overall positive compliance of RT-LAMP and RT-qPCR is 91%; for nasal swabs, when Ct < 30, the consistency of RT-LAMP and RT-qPCR is the same; for saliva samples, the positive detection rate of RT-LAMP was identical to that of RT-qPCR when Ct < 30.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (7)
1. An RT-LAMP primer and probe combination for detecting a new coronavirus, characterized in that the RT-LAMP primer and probe combination comprises one or more of a primer and probe for detecting an N gene of the new coronavirus, a primer and probe for detecting an E gene of the new coronavirus, and a primer and probe for detecting an ORF1A gene of the new coronavirus;
the primer sequences of the primer and the probe of the N gene are shown as SEQ ID No. 1-4, and the probe sequences are shown as SEQ ID No. 5;
the primer sequences of the primer and the probe of the E gene are shown in SEQ ID No. 6-9, and the probe sequences are shown in SEQ ID No. 10;
the primer sequences of the primer and the probe of the ORF1A gene are shown as SEQ ID No. 11-14, and the probe sequence is shown as SEQ ID No. 15.
2. Use of a primer and probe combination according to claim 1 for the preparation of a kit for detecting a novel coronavirus.
3. A kit for detecting a novel coronavirus based on RT-LAMP technology, comprising the primer and probe combination of claim 1.
4. The kit of claim 3, further comprising an RNA extraction reagent.
5. The kit according to claim 4, wherein the RNA extraction reagent comprises a lysate comprising TCEP-HCL 0.5-1 mg/. Mu.L, 0.4-0.6 MEDTA and guanidine hydrochloride 150-250 mM, pH < 9.
6. The kit of any one of claims 3 to 5, further comprising a positive control and a negative control, wherein the positive control is a new coronavirus and the negative control is DEPC water.
7. The kit according to any one of claims 3 to 5, wherein the sample of the novel coronavirus used in the detection of the kit is derived from saliva, a nasal or nasopharyngeal swab.
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| CN202310540273.4A CN117265180A (en) | 2023-05-15 | 2023-05-15 | RT-LAMP primer and probe combination for detecting novel coronavirus, kit and application of kit |
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| CN202310540273.4A CN117265180A (en) | 2023-05-15 | 2023-05-15 | RT-LAMP primer and probe combination for detecting novel coronavirus, kit and application of kit |
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