CN114540552A - Mosquito-borne flavivirus specificity qPCR detection method - Google Patents
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Abstract
The invention discloses a mosquito-borne flavivirus genus specificity qPCR detection method, belonging to the technical field of molecular biology and nucleic acid detection. The invention discloses a primer group for detecting mosquito-borne flavivirus, which comprises a nucleotide sequence shown as SEQ ID NO: 1 and the sequence shown in SEQ ID NO: 2, or a reverse primer as shown in figure 2. The primer group is designed and obtained according to the conserved regions of 12 mosquito-borne flavivirus genes, has good practical value for the amplification of various mosquito-borne flavivirus genes, realizes the simultaneous qualitative detection of 12 mosquito-borne flaviviruses by using 1 pair of primers, greatly reduces the detection cost, improves the detection flux and provides an effective new tool for the rapid and efficient detection of mosquito-borne flavivirus.
Description
Technical Field
The invention relates to the technical field of molecular biology and nucleic acid detection, in particular to a mosquito-borne flavivirus genus specificity qPCR detection method.
Background
Flaviviridae family Flaviviridae genus arboviruses include Yellow Fever Virus (YFV), Zika virus (ZIKV), Japanese Encephalitis Virus (JEV), dengue virus (DENV), West Nile Virus (WNV), Tembia virus (TBV), Bagaza virus (BGV), Israel turkey meningitis virus (ITV), Usturtia virus (UVSV), Stewart Venezhina virus (Sodwana virus, SPONV), Ileli virus (Ileuus virus, HV), Rocivirus (ROCIVIRU, ROVIRU), etc. These viruses are transmitted by mosquito bites and cause various diseases such as fever, rash, bleeding of skin mucous membranes, encephalitis, newborn malformations, etc., wherein DENV is widely prevalent in tropical and subtropical regions worldwide, particularly over 100 countries and regions in southeast asia, western pacific, central and south america and africa; YFV is mainly epidemic in Africa, but its spread is expanding in recent years, and Asia has been reported with many input cases; ZIKV is prevalent mainly in africa and south america, but many outbreaks have occurred in southeast asia; the prevalence of WNV also expands from north america to asia, europe; moreover, JEV is predominantly prevalent in asian areas, but in the region of approximately 7 million infected individuals per year, several other viruses have been rarely reported because they have not been routinely tested. The clinical characteristics of the diseases are highly similar, the clinical treatment mainly comprises symptomatic support treatment, and the prevention and control mainly comprises mosquito prevention and killing, so that even if species-specific detection of the pathogens cannot be carried out or is not required, a method which can simultaneously carry out species-specific universal detection on the pathogens also greatly facilitates the prevention, control and treatment of the diseases. In addition, in view of the need of disease prevention and control, the health and health departments in various regions need to monitor pathogens of these diseases on a large scale every year, including detection of pathogens in serum and medium of patients with fever of unknown causes, and a method capable of simultaneously detecting the above pathogens is also a necessary demand for improving detection throughput and saving manpower and material resources.
In the prior art, the detection method of the pathogen mainly comprises virus isolation culture, enzyme linked immunosorbent assay and PCR (polymerase chain reaction) detection.
The virus culture and isolation technology uses the collected sample to be cultured with cells, and determines whether the sample is infected by virus by observing cytopathic effect or detecting amplification of virus by other methods. Although the method is the gold standard for detecting the virus infection, the method can only detect virus particles with infection capacity in a sample, cannot determine the type of a pathogen, and needs PCR amplification or sequencing subsequently, and the virus isolation and culture operation procedure is complex, has biological safety level requirements on an experimental site and equipment, is long in detection time consumption, cannot be used for field detection, and even cannot be used for routine monitoring of large-scale samples.
Enzyme-linked immunosorbent assay is enzyme-labeled assay based on an antibody, and direct and indirect fluorescent assays and colloidal gold rapid assays utilize specific binding between an antigen and the antibody, and realize visual detection of pathogens by coupling the antibody with a chromogenic group. The antibody detection does not depend on large-scale equipment, and the detection result can be obtained within 30 minutes. However, the antibody detection has low sensitivity because of no signal amplification process, and false positive and false negative are easy to occur in the result. Due to the long preparation period of the new antibody, the method is difficult to be used for detecting the new virus, and the detection capability of the new virus subtype generated by antigen variation is doubtful.
The PCR detection technology is a process of visualizing products by using methods such as electrophoresis, fluorescence and the like after nucleic acid molecules of pathogens are amplified by polymerase, thereby realizing the purpose of detecting/monitoring the pathogens. After 1 or more rounds of nucleic acid amplification, the common PCR based on electrophoretic imaging still needs gel electrophoresis and imaging detection, takes long time, needs additional gel imaging equipment, and is not suitable for large-scale sample rapid detection on site. The fluorescent quantitative PCR is to directly read the result by detecting and collecting the fluorescent signal, and has the advantages of simple operation, high speed, high sensitivity, good specificity and the like. The fluorescent quantitative PCR can be divided into a probe method and a dye method, the probe method realizes the visualization of a detection result by adding an additional fluorescent probe, the specificity is higher, but the detection cost is correspondingly increased, the multiplex detection is limited by a fluorescent channel of a PCR instrument (generally 3/4 at present), and the disposable detection of all the viruses cannot be realized; the dye method does not need to additionally use a fluorescent probe, only 1 pair of primers can be matched with the fluorescent dye to detect the target, the amplification result can be interpreted through the amplification reaction and the melting reaction, the detection cost is low compared with the probe method, but the multiple pathogen specificity detection cannot be carried out. There is no report of high throughput detection of multiple mosquito-borne flaviviruses by dye methods.
Therefore, there is an urgent need in the art to develop a rapid, simple, efficient, and high-throughput assay for the above pathogens simultaneously.
Disclosure of Invention
The invention aims to provide a mosquito-borne flavivirus genus specificity qPCR detection method to solve the problems in the prior art, and the detection method simultaneously qualitatively detects 12 mosquito-borne flaviviruses through 1 pair of primers, greatly reduces the detection cost and improves the detection flux.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a primer group for detecting mosquito-borne flavivirus, which comprises a nucleotide sequence shown as SEQ ID NO: 1 and the sequence shown in SEQ ID NO: 2, or a reverse primer as shown in figure 2.
The invention also provides a kit for detecting the mosquito-borne flavivirus, which comprises the primer group.
The invention also provides a qPCR method for detecting the mosquito-borne flavivirus, which comprises the following steps:
(1) taking cDNA of a sample to be detected as a template;
(2) performing PCR amplification by using the primer group to obtain an amplification product;
(3) and analyzing the melting curve of the amplification product, and judging the type of the sample to be detected.
Further, in the step (2), the PCR amplification reaction system comprises the following components: 10 mul of 2XPCR buffer, 400nM concentration of upstream primer and downstream primer, 0.8 mul of each primer,cDNA template 1. mu.l, ddH2Make up to 20. mu.l of O.
Further, in the step (2), the PCR amplification reaction procedure is: 90s at 95 ℃ and 1 cycle; 5s at 95 ℃, 15s at 51 ℃ and 30s at 72 ℃ for 35 cycles.
Further, in step (3), the melting curve analysis program is: the temperature is between 65 and 95 ℃, the temperature is increased by 0.5 ℃ every 0.05 second, and the fluorescence is continuously collected.
Furthermore, if the melting curve has a specific melting peak, the sample to be detected can be judged to be the mosquito-borne flavivirus virus.
The invention also provides application of the primer group or the kit in preparation of products for detecting mosquito-borne flavivirus virus infection.
The invention discloses the following technical effects:
the invention obtains 12 mosquito-borne flavivirus gene sequences from a gene bank, obtains conserved regions of various mosquito-borne flavivirus genes through multiple sequence comparison and analysis, designs a pair of universal primers according to the conserved regions, optimizes PCR amplification reaction conditions through experiments, and performs virus detection according to PCR amplification reaction and melting curve analysis, thereby realizing simultaneous qualitative detection of 12 mosquito-borne flaviviruses by using 1 pair of primers, greatly reducing detection cost, improving detection flux, providing an effective new tool for rapid and efficient detection of mosquito-borne flavivirus viruses, greatly improving detection work efficiency of mosquito-borne flavivirus, being suitable for large-scale popularization and implementation, and having huge application potential and potential commercial value.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 shows the result of PCR amplification of yellow fever virus, wherein A is the amplification curve; b is a melting curve; c is a specific melting peak of the yellow fever virus;
FIG. 2 shows the PCR amplification result of Zika virus, wherein A is an amplification curve; b is a melting curve; c is a Zika virus specific melting peak;
FIG. 3 shows the amplification result of Japanese encephalitis virus PCR, wherein A is the amplification curve; b is a melting curve; c is encephalitis B virus specific melting peak;
FIG. 4 shows the result of PCR amplification of dengue virus type 1, wherein A is the amplification curve; b is a melting curve; c is a dengue virus type 1 specific melting peak;
FIG. 5 shows the result of PCR amplification of dengue virus type 2, wherein A is the amplification curve; b is a melting curve; c is a dengue virus type 2 specific melting peak;
FIG. 6 shows the result of PCR amplification of dengue virus type 3, wherein A is an amplification curve; b is a melting curve; c is a dengue virus type 3 specific melting peak;
FIG. 7 shows the result of PCR amplification of dengue virus type 4, wherein A is the amplification curve; b is a melting curve; c is a dengue virus type 4 specific melting peak;
FIG. 8 shows the PCR amplification result of West Nile virus, wherein A is the amplification curve; b is a melting curve; c is a specific melting peak of the West Nile virus;
FIG. 9 shows the result of PCR amplification of Tembusu virus, wherein A is the amplification curve; b is a melting curve; c is a specific melting peak of the tembusu virus;
FIG. 10 shows the result of PCR amplification of Baggera virus, wherein A is the amplification curve; b is a melting curve; c is a specific melting peak of the Baggeza virus;
FIG. 11 shows the result of PCR amplification of meningitis virus of Israel turkeys, where A is the amplification curve; b is a melting curve; c is a specific melting peak of the Israeli turkey meningitis virus;
FIG. 12 shows the result of PCR amplification of Usu soil virus, wherein A is an amplification curve; b is a melting curve; c is a specific melting peak of the ursolic soil virus;
FIG. 13 shows the result of PCR amplification of Spongwenni virus, in which A is an amplification curve; b is a melting curve; c is a specific melting peak of the Spongedwenni virus;
FIG. 14 shows the results of PCR amplification of Ilius virus, wherein A is the amplification curve; b is a melting curve; c is an illius virus specific melting peak;
FIG. 15 shows the PCR amplification results of Roxiella virus, wherein A is an amplification curve in a standard sigmoid shape; b is a melting curve; c is a Roxiella virus specific melting peak;
FIG. 16 is a no template negative control (NTC), wherein A is an amplification curve; b is a melting curve; c is a melting peak diagram;
FIG. 17 shows the verification results of the present invention on two dengue confirmed sera, wherein A is a sigmoid amplification curve, B is a melting curve, and C is a dengue virus amplification product specific melting peak.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
Example 1 mosquito-borne flavivirus genus specific qPCR detection method
1. Primer design and screening
Through genome length screening, 762 total sequences of YFV, ZIKV, JEV, DENV, WNV, TBV, BGV, ITV, UVSV, SPONV, ILHV and ROCV with genome length exceeding 100600nt in GenBank are obtained, multiple sequence alignment and analysis are carried out, a common conserved region of all viruses is searched, and two positions of the NS5 region of all the viruses are relatively conserved. The method specifically comprises the following steps: the 8666-9009 segment of YFV (NC-002031) has higher conservation and is suitable for being used as a primer design region; the segment 8684-9027 of ZIKV (NC-012523) has higher conservation and is suitable for being used as a primer design region; the section 8709-9052 of the JEV (NC-001437) has higher conservation and is suitable for being used as a primer design region; the segment 8697-8940 of DENV1(NC001477) has higher conservation and is suitable for being used as a primer design region; the conservation of the 8599-8942 segment of DENV2(KY937190) is high, and the primers are suitable for being used as primer design regions; the conservation of the 8585-8931 segment of DENV3(KY863456) is high, and the primers are suitable for being used as primer design regions; the segment 8589-8932 of DENV4(NC002640) has high conservation and is suitable for being used as a primer design region; the segment 8704-9047 of WNV (NC001563) has higher conservative property and is suitable for being used as a primer design region; the segment 8687-9030 of TBV (MN249640) has higher conservation and is suitable for being used as a primer design region; the segment 8687-9033 of BGV (NC012534) has higher conservation and is suitable for being used as a primer design region; the segment 8672-9027 of ITV (KC734550) has higher conservation and is suitable for being used as a primer design region; the conservation of the 8708-9051 segment of UVSV (MT863562) is higher, and the UVSV is suitable for being used as a primer design region; the section 8608-8951 of SPONV (NC029055) has higher conservation and is suitable for being used as a primer design region; the segment 8679-9031 of the ILHV (NC009028) has higher conservation, and is suitable for being used as a primer design region; the segment 8685-9028 of ROCV (NC040776) has high conservation and is suitable for being used as a primer design region. Intercepting the above-mentioned region from the comparison result, after the re-comparison, designing general primer. The method comprises the steps of adding a facultative base according to the principle that M is A/C, R, A/G, W, A/T, S, G/C, Y, C/T, K, G/T, V, A/G/C, H, A/C/T, D, A/G/T, B, G/C/T, N, A/G/C/T, and replacing N with I if the facultative property is larger than or equal to 12, wherein I is hypoxanthine. The designed primers are screened by using a reverse transcription of the holy next of Shanghai and a qPCR detection system to obtain the primers meeting the requirements, 2 primers are obtained by screening, and the sequences of the primers are shown in Table 1:
TABLE 1 primers for specific detection of mosquito-borne flaviviruses
| Primer name | Primer sequences (5 '-3') | Sequence identification |
| Fla-F1C | ATGACIGAYACIACICC(SEQ ID NO.1) | Seq 1 |
| Fla-R1C | TTBCCCATCATGTTRTAIA(SEQ ID NO.2) | |
2, PCR system establishment and reaction condition optimization
Selecting the gene sequences of the 12 virus cases, and obtaining the gene sequences from Shanghai organismsDouble-stranded DNA sequences were synthesized and after concentration and purity measurements using Nanodrop, units were converted to copies/ul. Diluting to 10% by diluting to avoid repeated freeze thawing3copies/ul and 104The copies/ul were split and stored at-80 ℃.
At 2.7 x 104YFV fragments of copies/ul were used as templates to establish the reaction system shown in Table 2.
TABLE 2 PCR reaction System
By optimizing the reaction conditions: primer usage, annealing temperature, annealing time, extension time, number of amplification cycles, five factors set up four horizontal orthogonal experiments, and 11 conditioning sets were designed as shown in table 3. The Real-time fluorescent quantitative detection system uses SYBR Green as a dye, and a channel for collecting fluorescence is set as SYBR Green I.
TABLE 3 combination of optimization conditions for amplification conditions
Analyzing the change process of the fluorescence amplification reaction in the reaction process, wherein the reaction is carried out at the fastest speed, the Cycle Time value (CT value) is the minimum, the NTC primer dimer-free condition combination is taken as the optimal combination, and finally: primer 400nM, reaction program 95 ℃ 90s, 1 cycle; 5s at 95 ℃, 15s at 51 ℃ and 30s at 72 ℃ for 35 cycles; namely, the reaction system is shown in table 4:
TABLE 4 reaction System
Melting curve analysis procedure is 65-95 deg.C, every 0.05 second raise 0.5 deg.C, continuous set fluorescence, and melting curve analysis is carried out.
FIGS. 1-15 are the PCR amplification results for YFV, ZIKV, JEV, DENV1, DENV2, DENV3, DENV4, WNV, TBV, BGV, ITV, UVSV, SPONV, ILHV, ROCV viruses, respectively, FIG. 16 is the PCR amplification result for the template-free negative control (NTC), where A is the amplification curve; b is a melting curve; c is a specific melting peak. Specifically, FIG. 1 shows the result of PCR amplification of yellow fever virus, wherein A is an amplification curve in a standard S-shape; b is a melting curve, and the fluorescence value is obviously reduced near 86 ℃ in the melting process; c is a yellow fever virus specific melting peak, the Tm value is 86 ℃, when A, B, C has the typical characteristics, and the NTC graph C has no melting peak at the corresponding Tm value position, and can judge that the yellow fever virus is amplified. FIG. 2 shows the PCR amplification result of Zika virus, wherein A is an amplification curve in a standard sigmoid form; b is a melting curve, and the fluorescence value is obviously reduced near 86.5 ℃ in the melting process; c is a Zika virus specific melting peak, Tm is 86.5 ℃, when A, B, C shows the above typical characteristics, and NTC graph C has no melting peak at the corresponding Tm, it can be judged that Zika virus is amplified. FIG. 3 shows the result of PCR amplification of Japanese encephalitis virus, wherein A is an amplification curve in a standard sigmoid shape; b is a melting curve, and the fluorescence value is obviously reduced near 86 ℃ in the melting process; c is encephalitis B virus specific melting peak, Tm is 86 ℃, when A, B, C has the above typical characteristics, and NTC graph C has no melting peak at the corresponding Tm position, it can be judged that there is encephalitis B virus amplification. FIG. 4 shows the result of PCR amplification of dengue virus type 1, wherein A is an amplification curve in a standard sigmoid shape; b is a melting curve, and a remarkable fluorescence value reduction occurs near 83.5 ℃ in the melting process; c is a dengue virus type 1 specific melting peak, Tm is 83.5 ℃, when A, B, C shows the above typical characteristics, and NTC graph C has no melting peak at the corresponding Tm, it can be judged that there is dengue virus type 1 amplification. FIG. 5 shows the result of PCR amplification of dengue virus type 2, wherein A is an amplification curve in a standard sigmoid shape; b is a melting curve, and a remarkable fluorescence value reduction occurs near 83 ℃ in the melting process; c is a dengue virus type 2 specific melting peak, Tm is 83 ℃, when A, B, C shows the above typical characteristics, and NTC diagram C has no melting peak at the corresponding Tm, it can be judged that there is dengue virus type 2 amplification. FIG. 6 shows the result of PCR amplification of dengue virus type 3, wherein A is an amplification curve in a standard sigmoid shape; b is a melting curve, and a remarkable fluorescence value reduction occurs near 84.5 ℃ in the melting process; c is a dengue virus type 3 specific melting peak, Tm is 84.5 ℃, when A, B, C shows the above typical characteristics, and NTC graph C has no melting peak at the corresponding Tm, it can be judged that there is dengue virus type 3 amplification. FIG. 7 shows the result of PCR amplification of dengue virus type 4, wherein A is an amplification curve in a standard sigmoid shape; b is a melting curve, and the fluorescence value is obviously reduced near 85 ℃ in the melting process; c is a dengue virus type 4 specific melting peak, Tm is 85 ℃, when A, B, C shows the above typical characteristics, and NTC graph C has no melting peak at the corresponding Tm, it can be judged that there is dengue virus type 4 amplification. FIG. 8 shows the PCR amplification result of West Nile virus, wherein A is the amplification curve in a standard S shape; b is a melting curve, and the fluorescence value is obviously reduced near 85 ℃ in the melting process; c is a specific melting peak of the West Nile virus, the Tm value is 85 ℃, when A, B, C shows the typical characteristics, and the NTC graph C has no melting peak at the corresponding Tm value position, the West Nile virus can be judged to be amplified. FIG. 9 shows the result of PCR amplification of Tembusu virus, wherein A is an amplification curve in a standard sigmoid form; b is a melting curve, and a remarkable fluorescence value reduction occurs near 84.5 ℃ in the melting process; c is a specific melting peak of the tembusu virus, the Tm value is 84.5 ℃, when A, B, C shows the typical characteristics, and the NTC graph C has no melting peak at the corresponding Tm value position, the tembusu virus is judged to be amplified. FIG. 10 shows the PCR amplification result of Baggera virus, wherein A is the amplification curve in a standard S-shape; b is a melting curve, and the fluorescence value is obviously reduced near 85 ℃ in the melting process; c is a melting peak specific to the Bagaza virus, the Tm value is 85 ℃, when A, B, C shows the typical characteristics, and the NTC graph C has no melting peak at the corresponding Tm value position, the amplification of the Bagaza virus can be judged. FIG. 11 shows PCR amplification results of the meningococcal virus of Israeli, in which A is an amplification curve in a standard S-shape; b is a melting curve, and the fluorescence value is obviously reduced near 86 ℃ in the melting process; c is specific melting peak of the Israeli turkey meningitis virus, Tm value is 86 ℃, when A, B, C shows the typical characteristics, and NTC graph C has no melting peak at the corresponding Tm value position, the Israeli turkey meningitis virus amplification can be judged. FIG. 12 shows the PCR amplification result of Usu soil virus, in which A is an amplification curve in a standard S-shape; b is a melting curve, and the fluorescence value is obviously reduced near 85 ℃ in the melting process; c is a fusion peak specific to the ursolic soil virus, the Tm value is 85 ℃, when A, B, C shows the typical characteristics, and simultaneously, when the NTC graph C has no fusion peak at the corresponding Tm value position, the amplification of the ursolic soil virus can be judged. FIG. 13 shows the result of PCR amplification of Spongwenni virus, in which A is an amplification curve in a standard sigmoid form; b is a melting curve, and a remarkable fluorescence value reduction occurs near 87 ℃ in the melting process; c is a Standy Wenyvirus specific melting peak, Tm is 87 ℃, when A, B, C shows the above typical characteristics, and NTC graph C has no melting peak at the corresponding Tm, it can be judged that there is amplification of Standy Wenyvirus. FIG. 14 shows the results of PCR amplification of Ilius virus, wherein A is the amplification curve in a standard sigmoid shape; b is a melting curve, and a remarkable fluorescence value reduction occurs near 87 ℃ in the melting process; c is an illius virus specific melting peak, Tm value is 87 ℃, when A, B, C shows the typical characteristics, and NTC graph C has no melting peak at the corresponding Tm value position, the illius virus amplification can be judged. FIG. 15 shows the PCR amplification results of Roxiella virus, wherein A is an amplification curve in a standard S-shape; b is a melting curve, and the fluorescence value is obviously reduced near 86.5 ℃ in the melting process; c is a specific melting peak of the Roxiella virus, the Tm value is 86.5 ℃, when A, B, C shows the typical characteristics, and the NTC graph C has no melting peak at the corresponding Tm value position, the Roxiella virus can be judged to be amplified. FIG. 16 is a no template negative control (NTC), where A is the amplification curve without sigmoid amplification; b is a melting curve, and no sharp fluorescence value decline curve exists in the melting process; c melting peak diagram, no melting peak.
Example 2 mosquito-borne flavivirus genus specific qPCR detection method specificity and reliability verification
After extracting nucleic acid from 2 and 92 dengue serum samples (all DENV3) which are diagnosed and excluded by commercial reagents (Calyday organisms, dengue virus RNA detection kit (PCR-fluorescent probe method), S1908-23Y) respectively, the samples are transcribed into cDNA by using Shanghai Seisan reverse transcription kit, and 1ul of cDNA is detected according to the qPCR amplification reaction and melting curve analysis method established in the example 1, the result is consistent with the detection result of the commercial kit, non-specific amplification and omission are avoided, and the result is reliable.
As can be seen from FIG. 17, both dengue serum samples have typical sigmoid amplification curves, the melting curve shows a significant fluorescence intensity decrease around 84.5 ℃, and the Tm value of the amplification product is 84.5 ℃, which is consistent with the melting peak of dengue virus type 3 in example 1, so the method has specific and reliable results in dengue virus detection.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Sequence listing
<110> prevention and cure for parasitic disease in Yunnan province
<120> mosquito-borne flavivirus genus specificity qPCR detection method
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (6)..(15)
<223> n = hypoxanthine
<400> 1
atgacngaya cnacncc 17
<210> 2
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
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Claims (8)
1. A primer set for detecting mosquito-borne flavivirus, wherein the primer set comprises a primer set with a nucleotide sequence shown in SEQ ID NO: 1 and the sequence shown in SEQ ID NO: 2, or a reverse primer as shown in figure 2.
2. A kit for detecting mosquito-borne flavivirus, said kit comprising the primer set of claim 1.
3. A qPCR method for detecting mosquito-borne flavivirus is characterized by comprising the following steps:
(1) taking cDNA of a sample to be detected as a template;
(2) performing PCR amplification by using the primer set according to claim 1 to obtain an amplification product;
(3) and (4) carrying out melting curve analysis on the amplification product, and judging the type of the sample to be detected.
4. The qPCR method for detecting mosquito-borne flavivirus of claim 3, wherein in step (2), the PCR amplification reaction system comprises the following components: 2XPCR buffer 10 ul, upstream and downstream primer concentrations are 400nM, each 0.8 ul, cDNA template 1ul, ddH2O make up to 20. mu.l.
5. The qPCR method for detecting mosquito-borne flavivirus of claim 3, wherein in step (2), the PCR amplification reaction procedure is as follows: 90s at 95 ℃ and 1 cycle; 5s at 95 ℃, 15s at 51 ℃ and 30s at 72 ℃ for 35 cycles.
6. The qPCR method for detecting mosquito-borne flavivirus of claim 3, wherein in step (3), the melting curve analysis procedure is: the temperature is between 65 and 95 ℃, the temperature is increased by 0.5 ℃ every 0.05 second, and the fluorescence is continuously collected.
7. The qPCR method for detecting mosquito-borne flavivirus of claim 3, wherein if the melting curve shows a specific melting peak, the sample to be detected can be determined to be mosquito-borne flavivirus.
8. Use of the primer set of claim 1 or the kit of claim 2 for the preparation of a product for detecting mosquito-borne flavivirus infection.
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| CN102643930A (en) * | 2012-04-05 | 2012-08-22 | 中华人民共和国大榭出入境检验检疫局 | Consensus-degenerate hybridoligonucleotide primer (CODEHOP) reverse transcription-polymerase chain reaction (RT-PCR) reagent and method for detecting flavivirus viruses |
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| CN102643930A (en) * | 2012-04-05 | 2012-08-22 | 中华人民共和国大榭出入境检验检疫局 | Consensus-degenerate hybridoligonucleotide primer (CODEHOP) reverse transcription-polymerase chain reaction (RT-PCR) reagent and method for detecting flavivirus viruses |
| CN108866238A (en) * | 2017-05-16 | 2018-11-23 | 北京义翘神州科技有限公司 | A kind of kit detecting a variety of arboviruses and invertebrate virus infection |
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