CN109207640B - Primer group, probe group and kit for detecting various respiratory viruses and application of primer group, probe group and kit - Google Patents
Primer group, probe group and kit for detecting various respiratory viruses and application of primer group, probe group and kit Download PDFInfo
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Abstract
The invention discloses a primer group for detecting various respiratory viruses, a probe group for detecting various respiratory viruses, a kit comprising the primer group and the probe group and application of the kit. The primer set and the probe set adopted by the invention have good specificity and high detection sensitivity, the primer set and the probe set are matched with a kit prepared from 2 xNASBA reaction liquid and 5 xNASBA reaction enzyme mixed liquid to amplify a sample to be detected, and then the fusion curve analysis technology is combined, so that the method is simple and convenient to operate, the result is easy to interpret, and different NASBA amplification products can be detected rapidly and accurately in real time.
Description
Technical Field
The invention relates to a primer group, a probe group and a kit for detecting various respiratory viruses and application thereof.
Background
Acute respiratory infections are a significant cause of morbidity and mortality in children worldwide. Pathogenic microorganisms responsible for acute respiratory infections are quite complex and include: bacteria, viruses, mycoplasma, chlamydia, fungi, etc., with viruses being most common. The symptoms caused by the infection of the respiratory viruses are not specific, and can not be diagnosed by clinical symptoms. At present, clinically common detection methods comprise a culture method, a biochemical method, an immune method and the like. The culture method has the advantages of complex operation, long detection period and high cost, and is not suitable for conventional detection of clinical samples. The biochemical detection method is simple and quick to operate and low in cost, but the method is low in sensitivity and specificity, has a plurality of influencing factors, and cannot distinguish specific types of viruses. The immunoassay method is rapid and simple, but one assay can only be directed against one of the viruses.
Nucleic acid sequence-dependent amplification (NASBA) technology, first reported by Guatelli et al in 1990, was mainly used for specifically amplifying RNA isothermally. In contrast, conventional PCR techniques only produce double replicons per cycle, whereas NASBA amplifies each timeRNA molecules are produced in 10-100 fold and can be produced up to 10 in 30 minutes 12 The amplification efficiency of the replicon is obviously higher than that of the common PCR. NASBA technology has been widely used to detect bacterial and viral infections including campylobacter, listeria, salmonella, HIV, HCV, avian influenza and the like. At present, most of internationally detecting NASBA amplification products adopts an open nucleic acid hybridization probe detection system, such as enzyme-linked color development or chemiluminescence for result evaluation, which has the defects and disadvantages of complex operation and easy generation of false positive and false negative due to amplification product pollution.
Disclosure of Invention
The invention aims to solve the problems that a nucleic acid hybridization probe detection system in the prior art is complex in operation and amplification product pollution is easy to generate false positive and false negative, and provides a primer group, a probe group and a kit for detecting various respiratory viruses and application thereof.
A primer set for detecting a plurality of respiratory viruses, the primer set comprising at least two primer pairs of:
a primer pair for influenza A virus, which consists of two sequences shown as SEQ ID NO.1 and SEQ ID NO.2 in a sequence table; a primer pair for influenza B virus, which consists of two sequences shown in SEQ ID NO.3 and SEQ ID NO.4 in a sequence table; a primer pair for parainfluenza virus type 1, which consists of two sequences shown in SEQ ID NO.5 and SEQ ID NO.6 in a sequence table; a primer pair for parainfluenza virus type 2, which consists of two sequences shown in SEQ ID NO.7 and SEQ ID NO.8 in a sequence table; a primer pair aiming at parainfluenza virus type 3, which consists of two sequences shown in SEQ ID NO.9 and SEQ ID NO.10 in a sequence table.
Preferably, the forward primers of the primer pair are each added at the 5' end with a T7RNA polymerase-recognized promoter sequence, which is TAACGACTCACTTAGAGG.
The invention also provides a probe set for detecting a plurality of respiratory viruses, wherein the probe set comprises at least 2 molecular beacon fluorescent probes as follows:
a molecular beacon fluorescent probe aiming at influenza A virus by a sequence shown in SEQ ID NO.12 in a sequence table; a molecular beacon fluorescent probe aiming at the influenza B virus by a sequence shown in SEQ ID NO.13 in the sequence table; a molecular beacon fluorescent probe aiming at parainfluenza virus type 1 by a sequence shown by SEQ ID NO.14 in a sequence table; a molecular beacon fluorescent probe aiming at parainfluenza virus type 2 by a sequence shown by SEQ ID NO.15 in a sequence table; the sequence shown by SEQ ID NO.16 in the sequence table aims at a parainfluenza virus type 3 molecular beacon fluorescent probe.
Preferably, both ends of each of the molecular beacon fluorescent probes are respectively provided with a fluorescent group and a quenching group.
Preferably, the fluorophore is selected from at least one of ALEX-350, FAM, VIC, TET, CAL, fluor Gold540, JOE, HEX, CAL Flourorange560, TAMRA, cal Fluor Red590, ROX, CAL Fluor, red610, TEXASRED, CAL FlourRed 635, quasar670, CY3, CY5, CY5.5 or Quasar705, and the quenching group is selected from one of DABCYL, BHQ, ECLIPSE or TAMRA.
The invention also provides a kit for detecting various respiratory viruses, which comprises the following reagents: (a) a primer set; (b) a set of probes.
Preferably, the kit further comprises a 2×nasba reaction solution, a 5×nasba reaction enzyme mixed solution.
Preferably, the 2 x NASBA reaction solution includes: tris-HCl, KCl, mgCl 2 dNTP, rNTP, DTT, betaine and DMSO; the 5 x NASBA reaction enzyme cocktail comprises: AMV reverse transcriptase, T7RNA polymerase, RNase H, RNase inhibitor and BSA.
Preferably, the solute solubility in the 2 x NASBA reaction solution is as follows: 150mM Tris-HCl,200mM KCl,30mM MgCl, pH8.3 2 1mM dNTP,4mM rNTP,20mM DTT,0.5mM betaine, DMSO at 20% volume fraction; the solute solubility in the 5×nasba enzyme cocktail is as follows: 1.6U/. Mu.L AMV reverse transcriptase, 10U/. Mu. L T7RNA polymerase, RNase H0.05U/. Mu.L, 3U/. Mu.L RNase inhibitor, 30mg/mL BSA.
The invention also provides an application of the kit for detecting various respiratory viruses, which uses RNA of a nasopharyngeal swab sample as a template, uses primer groups for amplifying two or more respiratory viruses and corresponding probe groups, and a mixed solution of 2 xNASBA reaction solution and 5 xNASBA reaction enzyme to carry out PCR reaction, and uses an amplified product as a sample to carry out melting curve analysis and detection.
The beneficial effects of the invention include:
1. the primer set and the probe set designed by the invention have good specificity and high detection sensitivity; the probe adopts multicolor fluorescent molecular beacon probe design, and can realize the differentiation and identification of different NASBA amplification products, thereby greatly improving the detection flux.
2. The kit provided by the invention can be used for simultaneously detecting various respiratory viruses.
3. The invention uses the specific primer group and the probe group to amplify the sample to be detected by matching with the 2 xNASBA reaction liquid and the 5 xNASBA reaction enzyme mixed liquid, and then combines the melting curve analysis technology to judge the existence of the amplified product, thereby having simple operation, easy interpretation of the result and high detection specificity.
Drawings
FIG. 1 is a graph showing fluorescence melting curves of influenza A virus in example 1 of the present invention.
FIG. 2 is a graph showing fluorescence melting curves of influenza B virus in example 1 of the present invention.
FIG. 3 is a graph showing the fluorescence melting curve of parainfluenza virus type 1 in example 1 of the present invention.
FIG. 4 is a graph showing the fluorescence melting curve of parainfluenza virus type 2 in example 1 of the present invention.
FIG. 5 is a graph showing the fluorescence melting curve of parainfluenza virus type 3 in example 1 of the present invention.
FIG. 6 is a plot of the fluorescence melting curve of the sensitivity of influenza B virus in example 2 of the present invention.
FIG. 7 is a graph showing the results of detecting influenza B virus and parainfluenza virus type 1 virus in a clinical sample according to example 3 of the present invention.
Detailed Description
The invention will be described in further detail with reference to the following detailed description and with reference to the accompanying drawings. It should be emphasized that the following description is merely exemplary in nature and is in no way intended to limit the scope of the invention or its applications.
The invention is used for detecting at least 2 of the following 5 respiratory viruses, which are respectively: influenza a virus, influenza b virus, parainfluenza virus type 1, parainfluenza virus type 2 and parainfluenza virus type 3.
The invention provides a kit for detecting various respiratory viruses, which comprises a primer group and a probe group; preferably, the kit further comprises a mixture of 2 XSBA reaction solution and 5 XSBA reaction enzyme; wherein the 2 XSBA reaction solution comprises Tris-HCl, KCl, mgCl 2 dNTP, rNTP, DTT, betaine and DMSO; the 5 XNASBA reaction enzyme mixture comprises: AMV reverse transcriptase, T7RNA polymerase, RNase H, RNase inhibitor and BSA.
Preferably, in some embodiments, the 2 XSBA reaction solution includes 150mM Tris-HCl (pH 8.3), 200mM KCl,30mM MgCl 2 1mM dNTP,4mM rNTP,20mM DTT,0.5mM betaine, DMSO at 20% volume fraction; the 5 XNASBA reaction enzyme mixture comprises: 1.6U/. Mu.L AMV reverse transcriptase, 10U/. Mu. L T7RNA polymerase, RNase H0.05U/. Mu.L, 3U/. Mu.L RNase inhibitor, 30mg/mL BSA.
In some embodiments, the primer set comprises at least two primer pairs having the sequences shown in table 1:
TABLE 1
Preferably, the forward primer of each virus is added with a promoter sequence capable of being recognized by T7RNA polymerase at the 5' end, wherein the promoter sequence is a sequence TATAACGACTCACTTAGAGG shown in SEQ ID No.11 in a sequence table.
In some embodiments, the probe set employs at least 2 of the following 5 molecular beacon fluorescent probes, the probe sequences are shown in table 2, the sequence numbers of the probes are SEQ ID No. 12-SEQ ID No.16, respectively:
TABLE 2
Each of the molecular beacon fluorescent probes described above carries a fluorophore and a quencher at each end, wherein in addition to the fluorophores FAM, ROX, CY, HEX carried in table 2 above, in other embodiments the fluorophores may be selected from ALEX-350, VIC, TET, CAL, fluor Gold540, JOE, CAL fluorescent orange560, TAMRA, CAL fluorescent Red590, CAL Fluor, red610, TEXASRED 635, quasar670, CY3, CY5.5, or Quasar705; in addition to the quenching group Dabcy1 as set forth in table 2 above, in other embodiments the quenching group may be selected from BHQ, ECLIPSE or TAMRA.
Preferably, the forward and reverse primers in the kit primer set are each 1. Mu.M, and the molecular beacon fluorescent probes are each 0.3. Mu.M.
Example 1. There is provided the use of a kit for detecting a plurality of respiratory viruses in the detection of respiratory viruses comprising the steps of:
(1) Preparation of primer set and probe set
Nucleic acid sequences of respiratory viruses are obtained from GenBank, homology comparison analysis is performed, conserved sequence regions of the viruses are determined, and proper sequences are selected in the conserved regions to design specific primer groups and probes. In particular, the primer set and probe may be designed according to the form of the target viral RNA to be detected.
(2) Preparing a kit: the kit consists of a primer group, a probe group and the mixed solution of the 2 xNASBA reaction solution and the 5 xNASBA reaction enzyme.
(3) Extraction of RNA: taking a positive virus sample, extracting RNA in the sample by using a commercial virus RNA extraction kit, wherein the specific operation is carried out according to the instruction.
(4) NASBA reaction system: taking 5 mu L of the RNA extracting solution of the virus, adding 12.5 mu L of mixed solution containing primer group, probe group and 2 xNASBA reaction solution, uniformly mixing, and adding 2.5 mu L of LDEPC treated water until the total reaction system is 20 mu L.
(5) NASBA amplification: heating the NASBA reaction system of the step (4) at 65 ℃ for 5min by using a PCR instrument, cooling at 41 ℃ for 5min, immediately adding 5 mu L of 5 XSBA reaction enzyme mixed solution, and reacting at 41 ℃ for 90min.
(6) NASBA amplification detection: performing melting curve analysis on the amplification product obtained in the step (5) on a fluorescence PCR instrument: heating at 95 ℃ for 2min, and denaturing the amplified product at high temperature to break double chains; cooling at 40 ℃ for 2min to enable the detection probe to hybridize with the target, and cooling to 40 ℃ conventionally; then, the temperature is increased from 40 ℃ to 85 ℃ at a heating rate of 0.1 ℃/s, melting curve analysis is carried out, and fluorescent signals are acquired at the stage.
(7) Analysis of results: judging the detection result according to the melting curve peak diagram, wherein the types of fluorescent channels and melting peak Tm values corresponding to each type of target virus are as follows:
| virus species | Fluorescence type | Melting peak Tm value |
| Influenza A virus | FAM | 65℃±0.5℃ |
| Influenza b virus | ROX | 58.5℃±0.5℃ |
| |
CY5 | 59℃±0.5 |
| Parainfluenza virus | ||
| 2 | HEX | 62℃±0.5 |
| Parainfluenza virus | ||
| 3 | CY5 | 55℃±0.5℃ |
The detection results of example 1 are shown in fig. 1 to 5, in which fig. 1 shows a fluorescence melting curve of influenza a virus, fig. 2 shows a fluorescence melting curve of influenza b virus, fig. 3 shows a fluorescence melting curve of parainfluenza virus type 1, fig. 4 shows a fluorescence melting curve of parainfluenza virus type 2, and fig. 5 shows a fluorescence melting curve of parainfluenza virus type 3.
The detection result shows that each type of target virus only detects melting curve peaks of specific Tm values in the corresponding fluorescent channels, and the detection kit has higher specificity; the kit provided by the invention can judge whether the amplified product exists or not by hybridizing the probe with the target and then denaturing the melting point of the melting point, the result is easy to judge, the error is not easy to occur, and the detection specificity is high; the NASBA technology and the molecular beacon melting curve analysis technology are combined, the specific probes are hybridized with corresponding target virus amplified products, and then the melting curve analysis is used for distinguishing various products, so that different NASBA amplified products can be identified, and the detection flux is greatly improved.
Example 2. Assay for detection sensitivity of a kit comprising the steps of:
(1) Preparation of RNA reference: the method comprises the steps of performing one-step RT-PCR (reverse transcription-polymerase chain reaction) by using various respiratory viral RNAs as templates and using an upstream primer with a promoter sequence capable of being recognized by T7RNA polymerase and a corresponding downstream primer, performing in-vitro transcription by using amplified product cDNA as a template according to the steps of Large Scale RNA Production System-T7 kit (Promega) instruction, extracting a transcription product with phenol and chloroform, precipitating isopropanol, dissolving with RNase-free water, quantifying for later use, sub-packaging and preserving at-70 ℃ to serve as an in-vitro transcribed RNA reference. To evaluate the detection sensitivity of the kit, the RNA reference was diluted 10-fold with TE buffer or DEPC treated water to prepare a sensitivity reference.
(2) NASBA amplification and NASBA amplification product detection were performed as in step (4) to step (7) of example 1.
Taking the sensitivity detection of the influenza b virus as an example, the detection result of example 2 is shown in fig. 6, the curve peak represents the melting peak detected by the reference substances with different concentrations from high to low, and the fluorescence derivative value of the melting peak represents the intensity of the fluorescence signal, so that the detection sensitivity of the kit is indirectly reflected.
Example 3 detection Effect of kit on clinical samples
(1) Extracting viral RNA: nasopharyngeal swab samples of suspected respiratory viral infected persons were collected and RNA was extracted from the samples using a commercial viral RNA extraction kit, and the specific procedures were as described.
(2) NASBA amplification and NASBA amplification product detection were performed as in step (4) to step (7) of example 1.
FIG. 7 shows the results of partial clinical samples, in which melting curve peak 1 is influenza B virus detected by ROX channel, melting curve peak 2 is parainfluenza virus type 1 virus detected by CY5 channel, and melting curve peak 3 is not detected by HEX channel, indicating that no virus is detected by this fluorescent channel.
Those skilled in the art will recognize that many variations are possible in light of the above description, and that the examples and figures are intended to be illustrative of one or more particular embodiments, and that any modifications, equivalents, and improvements made within the spirit and principles of the present invention are to be included within the scope of the present invention.
Sequence listing
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Claims (3)
1. An application of a primer group and a probe group in preparing a kit for detecting respiratory viruses, which is characterized in that the primer group is as follows:
a primer pair for influenza A virus, which consists of two sequences shown in SEQ ID NO.1 and SEQ ID NO. 2;
a primer pair for influenza B virus, which consists of two sequences shown in SEQ ID NO.3 and SEQ ID NO. 4;
a primer pair for parainfluenza virus type 1, consisting of two sequences shown in SEQ ID No.5 and SEQ ID No. 6;
a primer pair for parainfluenza virus type 2, consisting of two sequences shown in SEQ ID No.7 and SEQ ID No. 8;
a primer pair for parainfluenza virus type 3 consisting of two sequences shown in SEQ ID NO.9 and SEQ ID NO. 10;
the forward primers in the primer set are added with a promoter sequence which can be recognized by T7RNA polymerase at the 5' end, and the promoter sequence is TAACGACTCACTTAGAGG;
correspondingly, the probe group is as follows:
a molecular beacon fluorescent probe for influenza A virus, which consists of a sequence shown in SEQ ID NO.12, wherein the 5 'end of the molecular beacon fluorescent probe is provided with a FAM fluorescent group, and the 3' end of the molecular beacon fluorescent probe is provided with a Dabcy1 quenching group;
a molecular beacon fluorescent probe for influenza B virus, which consists of a sequence shown in SEQ ID NO.13, wherein the 5 'end of the molecular beacon fluorescent probe is provided with a ROX fluorescent group, and the 3' end of the molecular beacon fluorescent probe is provided with a Dabcy1 quenching group;
a molecular beacon fluorescent probe for parainfluenza virus type 1, which consists of a sequence shown in SEQ ID NO.14, wherein the 5 'end of the molecular beacon fluorescent probe is provided with a CY5 fluorescent group, and the 3' end of the molecular beacon fluorescent probe is provided with a Dabcy1 quenching group;
a molecular beacon fluorescent probe for parainfluenza virus type 2, which consists of a sequence shown in SEQ ID No.15, wherein the 5 'end of the molecular beacon fluorescent probe is provided with a HEX fluorescent group, and the 3' end of the molecular beacon fluorescent probe is provided with a Dabcy1 quenching group;
a molecular beacon fluorescent probe for parainfluenza virus type 3, which consists of a sequence shown in SEQ ID No.16, wherein the 5 'end of the molecular beacon fluorescent probe is provided with a CY5 fluorescent group, and the 3' end of the molecular beacon fluorescent probe is provided with a Dabcy1 quenching group;
wherein, the kit distinguishes each type of target virus through melting curve analysis, and the type of fluorescent channel and melting peak Tm value corresponding to each type of target virus are as follows:
2. the use of claim 1, wherein the kit comprises: tris-HCl, KCl, mgCl 2 dNTP, rNTP, DTT, betaine and DMSO, AMV reverse transcriptase, T7RNA polymerase, RNase H, RNase inhibitor and BSA.
3. The use according to claim 2, wherein the solute solubility in the kit is as follows: 150mM Tris-HCl,200mM KCl,30mM MgCl, pH8.3 2 ，1 mM dNTP，4 mM rNTP，20 mMDTT,0.5mM betaine, 20% DMSO by volume fraction, 1.6U/. Mu.L AMV reverse transcriptase, 10U/. Mu. L T7RNA polymerase, RNase H0.05U/. Mu.L, 3U/. Mu.L RNase inhibitor, 30mg/mL BSA.
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| CN111004867B (en) * | 2020-01-03 | 2020-12-18 | 牡丹江医学院 | Influenza A virus detection primer, probe and kit thereof |
| CN111826469A (en) * | 2020-08-18 | 2020-10-27 | 杭州昱鼎生物科技有限公司 | 2019-nCoV nucleic acid isothermal amplification primer based on molecular beacon, kit and detection method |
| CN112111566A (en) * | 2020-09-23 | 2020-12-22 | 迈克生物股份有限公司 | Multiplex nucleic acid detection method, combination and kit |
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