CN112593009A - Primer group for simultaneously detecting three mosquito-borne viruses based on capillary electrophoresis and application thereof - Google Patents
Primer group for simultaneously detecting three mosquito-borne viruses based on capillary electrophoresis and application thereof Download PDFInfo
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Abstract
The invention relates to a primer group for simultaneously detecting three mosquito-borne viruses based on capillary electrophoresis and application thereof, belonging to the technical field of detection of mosquito-borne transmitted viruses. The invention provides a primer group which comprises three pairs and is used for respectively and specifically detecting three mosquito-borne viruses DENV/YFV/ZIKV, wherein the lengths of corresponding detection target fragments are respectively 79bp, 139bp and 168 bp. Meanwhile, a molecular Marker for simultaneously detecting the three mosquito-borne viruses based on capillary electrophoresis, a sample to be detected treatment system, an RT-PCR reaction system, reaction conditions and result analysis are disclosed. The method can rapidly and simultaneously screen three mosquito-borne viruses DENV/YFV/ZIKV, has the outstanding advantages of high detection sensitivity, strong specificity, capability of realizing multiple rapid and accurate detection, simple operation, convenient application and the like, and provides a feasible technical method for rapid detection of pathogens in the fields of clinical diagnosis, disease monitoring detection, inspection and quarantine and the like.
Description
Technical Field
The invention relates to a primer group for simultaneously detecting three mosquito-borne viruses based on capillary electrophoresis and application thereof, belonging to the technical field of detection of mosquito-borne transmitted viruses.
Background
Flaviviridae (Flaviviridae) contains a large family of RNA families with single-stranded positive strands of their genomes, currently divided into four genera, flaviviruses (flaviviruses), pestiviruses (pestiviruses), hepaciviruses (hepaciviruses), and Pegivirus. Dengue virus (Dengue virus, DENV), Zika virus (ZIKV) and Yellow fever virus (Yellow fever virus, YFV) belong to Flaviviridae (Flaviviridae) members, and viruses have many similarities in particle morphology, genome structure, replication strategy and the like, but have different biological characteristics, and lack cross protection in serology among them, which increases certain difficulties in disease prevention and diagnosis. The three viruses are mainly prevalent in southeast Asia, Africa, America and the Western Pacific, and are transmitted by mosquito bites, and Aedes mosquito is the main transmission medium. With the increasing frequency of international trade, personnel and the like, the regional and international spread of mosquito-borne infectious diseases such as dengue fever, Zika and the like becomes a new normal state, and increasingly serious public health problems are caused. Zika virus, which has developed epidemic in America and caused infantile microcephaly and Guillain-Barre syndrome (GBS) in 2016, has attracted global attention as a sudden public health event, and there are many cases of input in China. The mosquito-borne infectious diseases such as ZIKV and DENV are continuously evolved from jungle circulation to urban circulation, so that tens of thousands of people are infected every year, and the life health of the people is seriously threatened. Early prevention is an important means for preventing and controlling outbreak of diseases, and the development of a rapid and accurate detection technology is a key for early prevention and control. With the continuous development of Capillary Electrophoresis (CE), the method is widely applied to the identification of food microorganisms and pathogens such as respiratory tract.
On the basis of systematic analysis of flavivirus, specific primer pairs are respectively designed for sequences in conserved regions of three common mosquito-borne virus infectious diseases including DENV, ZIKV and YFV, and target fragments with single base difference can be accurately judged by one-step RT-PCR combined with capillary electrophoresis, so that the single mosquito-borne virus can be quickly identified. By further constructing a molecular Marker based on capillary electrophoresis, the targets of simultaneously, rapidly and accurately identifying three mosquito-borne toxic diseases are realized. The rapid identification technical method which is established by the research and is based on capillary electrophoresis and simultaneously accurately identifies the three mosquito-borne viral diseases provides a new technical support for the defense and control of mosquito-borne infectious diseases of institutions such as national ports, primary medical treatment and the like.
Disclosure of Invention
The invention aims to overcome the defects of long time consumption, low detection efficiency, high detection cost, low detection timeliness and the like in the detection process of the mosquito-borne viruses, provides a primer group for simultaneously detecting three mosquito-borne viruses based on capillary electrophoresis and application thereof, and can simultaneously detect three mosquito-borne viruses DENV, YFV and ZIKV.
The technical scheme of the invention is that a primer group for simultaneously detecting three mosquito-borne viruses based on capillary electrophoresis comprises 3 primer pairs, namely a primer pair 1 aiming at mosquito-borne dengue virus DENV, a primer pair 2 aiming at mosquito-borne yellow fever virus YFV and a primer pair 3 aiming at mosquito-borne virus ZIKV; each primer pair comprises an upstream primer and a downstream primer.
Further specifically comprises an upstream primer F1 aiming at the DENV of the mosquito vector virus, the sequence of which is shown as SEQ ID No.1, and a downstream primer R1, the sequence of which is shown as SEQ ID No. 2;
an upstream primer F2 for the mosquito vector YFV has a sequence shown in SEQ ID No.3, and a downstream primer R2 has a sequence shown in SEQ ID No. 4;
the sequence of the upstream primer F3 for the ZIKV of the mosquito-borne virus is shown in SEQ ID No.5, and the sequence of the downstream primer R3 is shown in SEQ ID No. 6. Specifically, the following table 1 shows.
TABLE 1
A detection cassette comprising the primer set.
The application of the detection box simultaneously detects dengue virus DENV, yellow fever virus YFV and Zika virus ZIKV through PCR reaction.
Further, the application of the detection kit, the molecular marker adopted during PCR detection is specifically configured as follows by taking a 25 μ L reaction system as an example, and the final concentration of the used amount is as follows: reaction solution of Piper 2: 12.5 mu L; DNA polymerase: 0.5 mu L; RT reverse transcriptase: 0.5 mu L; an upstream primer: 1 mu L of the solution; a downstream primer: 1 mu L of the solution; viral template RNA: 5 mu L of the solution; ddH2O：4.5μL；Up to 25μL。
Furthermore, the concentrations of three pairs of primer pairs in the upstream primer and the downstream primer are consistent; wherein the total concentration of the upstream primer is 2.5 mu M, and the total concentration of the downstream primer is 2.5 mu M; the virus template RNA comprises three corresponding virus template RNAs, and the concentrations of the three are 10 ng/. mu.L.
In the PCR detection, a detection sample is specifically configured as follows by taking a 25 μ L reaction system as an example, and the final concentration of the usage amount is as follows: reaction solution of Piper 2: 12.5 mu L; DNA polymerase: 0.5 mu L; RT reverse transcriptase: 0.5 mu L; an upstream primer: 1 mu L of the solution; a downstream primer: 1 mu L of the solution; a sample to be detected: 5 mu L of the solution; ddH2O：4.5μL；Up to 25μL。
The application of the detection box is characterized in that: the PCR reaction procedure is shown in Table 2 below.
TABLE 2
The invention has the beneficial effects that: the invention can simultaneously detect three mosquito-borne viruses including DENV, YFV and ZIKV, has the outstanding advantages of high detection sensitivity, strong specificity, capability of realizing multiple rapid and accurate detection, simple operation, convenient application and the like, and provides a feasible technical method for rapid detection of pathogens in the fields of clinical diagnosis, disease monitoring detection, inspection and quarantine and the like.
Drawings
FIG. 1 is the signal spectrum of capillary electrophoresis of three mosquito-borne viruses.
FIG. 2 is the capillary electrophoresis gel imaging spectrum of three mosquito vector viruses.
FIG. 3 is the establishment of three mosquito vector virus capillary electrophoresis molecule markers.
FIG. 4 is a DENV/YFV/ZIKV capillary electrophoresis signal map.
FIG. 5 is a determination of the results of molecular marker versus DENV capillary electrophoresis.
FIG. 6 shows the result of YFV capillary electrophoresis judged by molecular marker.
FIG. 7 is a molecular marker versus judgment of the results of ZIKV capillary electrophoresis.
Detailed Description
Example 1
In the embodiment of the invention, the RT-PCR amplification reaction system for simultaneously detecting three mosquito-borne viruses DENV, YFV and ZIKV provided by the invention has the following reaction conditions and detection steps:
(1) sample preparation: the sample nucleic acid can be directly extracted from samples such as mosquito-borne tissue cells infected with viruses, blood and the like, can be extracted manually or by a full-automatic nucleic acid extractor, and the extracted nucleic acid is directly used for the next amplification reaction.
(2) Preferably, the One-step qRT-PCR Kit (TOYOBO) is selected and specifically configured as follows by taking a 25-mu L reaction system as an example:
the reaction system of 25. mu.L is specifically configured as follows, and the final concentration of the used amount is: reaction solution of Piper 2: 12.5 mu L; DNA polymerase: 0.5 mu L; RT reverse transcriptase: 0.5 mu L; an upstream primer: 1 mu L of the solution; a downstream primer: 1 mu L of the solution; viral template RNA: 5 mu L of the solution; ddH2O：4.5μL；Up to 25μL。
In the upstream primer and the downstream primer, the concentrations of three pairs of primer pairs are consistent (0.33 mu L is added to the upstream primer and the downstream primer of the three viruses respectively); wherein the total concentration of the upstream primer is 2.5 mu M, and the total concentration of the downstream primer is 2.5 mu M.
(3) Preferably, the PCR reaction procedure is shown in Table 3.
TABLE 3
(4) Analyzing and judging results:
the PCR amplification product was subjected to a full-automatic capillary electrophoresis apparatus (Qseq100, Yongding pot Biotechnology (Jiangsu) Ltd. (Su stock Co., Ltd. 20180095)), high-resolution clip S1(C105102) was selected, Alignment Marker MA1(C109100) was selected, Size Marker MA2(C109200) was selected, sample injection voltage was 4kv 10S, and separation pressure was 6kv 330S. The PCR amplification product was diluted 10-fold with Dilution Buffer (C104405) and subjected to capillary electrophoresis according to the instructions of the apparatus.
The capillary electrophoresis signal spectra of three mosquito-borne viruses are shown in figure 1, wherein A is electrophoresis Size marker, B is DENV, C is YFV, and D is ZIKV. The PCR products of the three mosquito-borne viruses can accurately judge a single target fragment (the error is not more than 3bp) with expected size after capillary electrophoresis, and the PCR products have better specificity, detection sensitivity and accuracy.
The capillary electrophoresis gel imaging spectra of the three mosquito-borne viruses are shown in figure 2, wherein the PCR amplification products of the three mosquito-borne viruses can amplify specific bands after electrophoresis.
The three capillary electrophoresis molecule markers of mosquito vector virus are constructed as shown in FIG. 3 (prepared according to 25. mu.L of the reaction system in example 1: 12.5. mu.L of the PIKHz 2 reaction solution, 0.5. mu.L of DNA polymerase, 0.5. mu.L of RT reverse transcriptase, 1. mu.L of the mixture of three viral upstream primers, 1. mu.L of the mixture of downstream primers, 5. mu.L of RNA containing three viral templates, and ddH2O：4.5μL；Up to 25μL。
In the upstream primer and the downstream primer, the concentrations of three pairs of primer pairs are consistent (0.33 mu L is added to the upstream primer and the downstream primer of the three viruses respectively); wherein the total concentration of the upstream primer is 2.5 mu M, and the total concentration of the downstream primer is 2.5 mu M. The total concentration of the RNA of the three virus templates is 10 ng/. mu.L), the deviation of the interpretation signal of the capillary electrophoresis target gene is not more than 3bp due to the difference of the linear structures of different target genes, and the confidence intervals of the interpretation signal of the three mosquito-borne virus target genes of DENV, YFV and ZIKV are respectively set at 78 (+/-3) bp, 139 (+/-3) bp and 169 (+/-3) bp according to experimental data. By establishing the molecular marker for the three mosquito-borne viruses, the target genes of the three mosquito-borne viruses can be synchronously and accurately detected.
Example 2
The results of nucleic acid extraction, RT-PCR reaction configuration and capillary electrophoresis analysis using DENV, YFV and ZIKV positive samples as detection targets and the established molecular marker as reference as described in example 1 (0.33. mu.L (final concentration: 0.25. mu.M) for the upstream and downstream primers of the three viruses, respectively) are shown below (FIG. 4): in FIG. 4, the molecular Marker is used as a reference, and the corresponding target fragment can be accurately detected after nucleic acid extraction, PCR amplification and electrophoresis, so that the purpose of simultaneously and accurately identifying the three mosquito-borne viruses is achieved.
The judgment of the molecular marker on the DENV capillary electrophoresis result is shown in FIG. 5, the judgment of the molecular marker on the YFV capillary electrophoresis result is shown in FIG. 6, and the judgment of the molecular marker on the ZIKV capillary electrophoresis result is shown in FIG. 7. Wherein when the target peak interval is 79(+3/-3), DENV is determined to be positive; YEV positive when the target peak interval is 139(+ 3/-3); when the target peak interval is 169(+3/-3), ZIKV positivity is determined.
The invention has unique innovation in the market, namely realizes the simultaneous detection of three mosquito-borne viruses, has the outstanding advantages of accurate detection, strong specificity, high sensitivity, quick detection time and the like, and avoids the defects of long time consumption, high detection cost, easy cross contamination and the like caused by one-by-one detection. Similar related technologies and product reports are not found in the market at present, and the invention has wide application prospects in the aspects of disease detection, infectious disease prevention and control, guidance of clinical treatment and the like.
The foregoing shows and describes the general principles, principal features, and advantages of the invention. It will be understood by those skilled in the art that the invention is not limited to the embodiments described above, which are merely illustrative of the principles of the invention, but that various changes and modifications may be made without departing from the spirit and scope of the invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Sequence listing
<110> Wuxi customs of the people's republic of China
Xinjiang Huanyuan environmental protection science and technology Limited company
Xinjiang international travel health care center (Wulu wood Qi customs port outpatient department)
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Claims (8)
1. A primer group for simultaneously detecting three mosquito-borne viruses based on capillary electrophoresis is characterized in that: comprises 3 groups of primer pairs, namely a primer pair 1 aiming at the mosquito-borne dengue virus DENV, a primer pair 2 aiming at the mosquito-borne yellow fever virus YFV and a primer pair 3 aiming at the mosquito-borne virus ZIKV; each primer pair comprises an upstream primer and a downstream primer.
2. The primer group for simultaneously detecting three mosquito-borne viruses based on capillary electrophoresis as claimed in claim 1, which is characterized in that: the primer specifically comprises an upstream primer F1 for the DENV of the mosquito vector virus, the sequence of which is shown in SEQ ID No.1, and a downstream primer R1, the sequence of which is shown in SEQ ID No. 2;
an upstream primer F2 for the mosquito vector YFV has a sequence shown in SEQ ID No.3, and a downstream primer R2 has a sequence shown in SEQ ID No. 4;
the sequence of the upstream primer F3 for the ZIKV of the mosquito-borne virus is shown in SEQ ID No.5, and the sequence of the downstream primer R3 is shown in SEQ ID No. 6.
3. A kit comprising the primer set according to claim 1 or 2.
4. Use of a cartridge according to claim 3, wherein: dengue virus DENV, yellow fever virus YFV and Zika virus ZIKV are simultaneously detected by PCR reaction.
5. Use of a cartridge according to claim 3, wherein: during PCR detection, the adopted molecular marker is specifically configured as follows by taking a 25 mu L reaction system as an example, and the final concentration of the used amount is as follows: reaction solution of Piper 2: 12.5 mu L; DNA polymerase: 0.5 mu L; RT reverse transcriptase: 0.5 mu L; an upstream primer: 1 mu L of the solution; a downstream primer: 1 mu L of the solution; viral template RNA: 5 mu L of the solution; ddH2O：4.5μL；Up to 25μL。
6. Use of a cartridge according to claim 3, wherein: the concentrations of three pairs of primer pairs in the upstream primer and the downstream primer are consistent; wherein the total concentration of the upstream primer is 2.5 mu M, and the total concentration of the downstream primer is 2.5 mu M; the virus template RNA comprises three corresponding virus template RNAs, and the concentrations of the three are 10 ng/. mu.L.
7. Use of a cartridge according to claim 3, wherein: in the PCR detection, a detection sample is specifically configured as follows by taking a 25 μ L reaction system as an example, and the final concentration of the usage amount is as follows: reaction solution of Piper 2: 12.5 mu L; DNA polymerase: 0.5 mu L; RT reverse transcriptase: 0.5 mu L; an upstream primer: 1 mu L of the solution; a downstream primer: 1 mu L of the solution; a sample to be detected: 5 mu L of the solution; ddH2O：4.5μL；Up to 25μL。
8. Use of a cartridge according to claim 3, wherein: the PCR reaction procedure was as follows:
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103911463A (en) * | 2014-04-03 | 2014-07-09 | 河北国际旅行卫生保健中心 | Kit for detecting mosquito borne pathogens and detection method thereof |
| CN110066889A (en) * | 2019-05-21 | 2019-07-30 | 四川国际旅行卫生保健中心 | Quick detection primer group, kit and its application of the GeXP of four kinds of Flavivirus virus are detected simultaneously |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103911463A (en) * | 2014-04-03 | 2014-07-09 | 河北国际旅行卫生保健中心 | Kit for detecting mosquito borne pathogens and detection method thereof |
| CN110066889A (en) * | 2019-05-21 | 2019-07-30 | 四川国际旅行卫生保健中心 | Quick detection primer group, kit and its application of the GeXP of four kinds of Flavivirus virus are detected simultaneously |
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