CN107988429B - Reagent for detecting rabies virus and application thereof - Google Patents
Reagent for detecting rabies virus and application thereof Download PDFInfo
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- CN107988429B CN107988429B CN201711205134.7A CN201711205134A CN107988429B CN 107988429 B CN107988429 B CN 107988429B CN 201711205134 A CN201711205134 A CN 201711205134A CN 107988429 B CN107988429 B CN 107988429B
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Abstract
The invention relates to the technical field of veterinary medicine pathogenic microorganism detection, and discloses a reagent for detecting rabies viruses and application thereof. The reagent consists of an upstream primer of a nucleotide sequence shown in SEQ ID NO. 1, a downstream primer of a nucleotide sequence shown in SEQ ID NO. 2 and a probe of a nucleotide sequence shown in SEQ ID NO. 3. The invention provides a group of primers and probe reagents suitable for RPA, which can accurately detect rabies viruses, have high specificity, sensitivity and accuracy, and make up for the defect that the existing RPA detection technology lacks detection reagents with excellent effects.
Description
Technical Field
The invention relates to the technical field of veterinary medicine pathogenic microorganism detection, and particularly relates to a reagent for detecting rabies viruses and application thereof.
Background
Rabies Virus (RV) belongs to the Rhabdoviridae (Rhabdoviridae) genus Lyssavirus (Lyssavirus). The shape is elastic, the nucleocapsid is spirally symmetrical, the surface is provided with a coating, and the single-stranded RNA is contained in the coating. Is the causative agent of rabies. Rabies has a wide epidemic range and is reported in almost all areas of the world. 99% of patients who die due to rabies are distributed in africa and asia, of which 56% are 44% in africa. China is a severe epidemic area of rabies, and the number of diseases is on the rise in recent years, so that a detection technology for quickly determining the disease is urgently needed, and a new method is provided for field experimental diagnosis and epidemiological investigation of rabies.
At present, the main method for detecting virus nucleic acid is a PCR method, and the conventional PCR detection needs a special thermal cycler and a complicated test procedure and is difficult to meet the detection requirements under field or field conditions. In recent years, isothermal amplification techniques for nucleic acids have been developed, which do not require an expensive PCR instrument, can rapidly amplify a large amount of target fragments in a short time, and have the advantages of simplicity, rapidity, high sensitivity, and the like. Among them, the RPA technique is more referred to as a nucleic acid detection technique that can replace PCR.
RPA technology relies primarily on three enzymes: recombinases that bind single-stranded nucleic acids (oligonucleotide primers), single-stranded DNA binding proteins (SSBs), and strand-displacing DNA polymerases. The mixture of these three enzymes is also active at ambient temperature, with an optimum reaction temperature around 37 ℃. RPA technology has been developed based on the principle of recombinase polymerase-mediated amplification, which mimics DNA replication in organisms. The method can increase the target gene exponentially in a very short time, and can realize real-time monitoring of template amplification if matched with a fluorescence-labeled probe and a fluorescence signal detector. The RPA technology greatly reduces the detection time, simplifies the reaction procedure, is very suitable for field or field detection after combining the rapid nucleic acid extraction technology and the portable real-time fluorescence detection equipment, and has wide application prospect. The method for detecting the rabies viruses by utilizing the RPA technology has great clinical application value.
The key to the analysis of RPA is the design of amplification primers and probes, which are not as mature as traditional PCR, and PCR primers are mostly not suitable for RPA. The design principle of the RPA primer generally comprises the following points:
(1) 3 to 5 nucleotides at the 5' end avoid polyguanines, preferably cytosines, which promote recombination; (2) the 3' end 3 nucleotides are G or C, which contributes to the stability of polymerase; (3) no polypurine and no pyrimidine are present; (4) the GC content is 30-70% to avoid forming a secondary structure, a hairpin structure and the like; (5) the target area avoids the repetitive elements.
Because no primer design software aiming at RPA exists at present, the primer design principle is only a general design principle, and if a better amplification effect, such as specificity and sensitivity, needs to be obtained, technicians need to manually search conditions for design optimization.
Disclosure of Invention
In view of the above, the present invention aims to provide a reagent for detecting rabies viruses, which is suitable for RPA and has high specificity, sensitivity and accuracy;
the invention also aims to provide application of the reagent in preparing a rabies virus detection kit.
In order to achieve the above purpose, the invention provides the following technical scheme:
a reagent for detecting rabies viruses comprises an upstream primer of a nucleotide sequence shown by SEQ ID NO. 1, a downstream primer of a nucleotide sequence shown by SEQ ID NO. 2 and a probe of a nucleotide sequence shown by SEQ ID NO. 3.
In a specific embodiment of the invention, the sequences of the primers and probes in the reagent are as follows:
upstream primer CTCTGGTGGAGATAAAACGTACTGATGTAG (SEQ ID NO: 1);
downstream primer CTCAACCTATACAGACTCAAGAGAAGACCG (SEQ ID NO: 2);
probe AGGCATGGAACTGACAAGAGACCCCACTG (BHQ1-dT) C (THF) C (FAM-dT) GAGCATGCGTCCTTA (SEQ ID NO: 3);
wherein BHQ1-dT represents thymine deoxynucleotide carrying fluorescence quenching group BHQ1, THF represents tetrahydrofuran linker, FAM-dT represents thymine deoxynucleotide carrying fluorescein group FAM.
The reagent of the invention is used for rapid detection, nucleic acid RNAs of rabies virus CVS-11 strains, JX-08-45 strains and BD06 strains separated in China are taken as templates to obtain obvious amplification curves, and other pathogenic nucleic acids such as canine distemper virus nucleic acid, canine coronavirus nucleic acid, rotavirus nucleic acid, canine parvovirus nucleic acid and total nucleic acid of healthy canine pharyngeal swab are taken as templates to carry out RPA reaction without amplification curves.
In addition, in the sensitivity test, viral nucleic acid was diluted 10-fold to multiple gradients, resulting in a detection limit of 102And the sensitivity is high. Meanwhile, detecting 12 samples in the domestic capability verification test samples; nucleic acid templates in samples to be tested were extracted using QIAGEN RNeasy Mini Kit from Qiagen, and then the samples were tested for rabies virus by the RPA method, and 12 samples were tested in parallel by the general RT-PCR method recommended by the world animal health Organization (OIE). Results the two detection methods have consistent detection results. The results show that the results obtained by the primer group and the probe in the reagent and the detection method thereof are consistent with the results of RT-PCR, and the reliability of the invention is proved.
Based on the excellent technical effects, the invention provides the application of the reagent in preparing the rabies virus detection kit. Among them, in the present embodiment, the rabies viruses are the CVS-11 strain rabies virus and the BD06 strain rabies virus.
According to the application, the invention provides a kit for detecting rabies viruses, which comprises the reagent.
To further refine the kit, the kit can also comprise a rabies virus RNA extraction reagent and an RPA amplification reaction reagent.
The rabies virus RNA extraction reagent can comprise one or more of reagents which are conventionally used for extracting rabies virus strain RNA in the field, and in the specific embodiment of the invention, the rabies virus RNA extraction reagent adopts various reagents in a High Pure PCR Template Preparation kit of Roche company.
The RPA amplification reaction reagents may include one or more of the reagents conventional in the art for RPA reaction systems, such as rehydration buffer, DEPC water, RPA lyopilization beads, magnesium acetate solution, etc. in a twist amp exo RT (twist dx, Cambridge, UK) kit.
In the specific embodiment of the invention, the RPA reaction system (50 μ l in total) is reacted for 15 minutes at 37 ℃ in an RPA amplification detector or a fluorescence quantitative PCR instrument, and the system comprises the following components:
rehydrating buffer solution 29.5 μ l, upstream and downstream primers of 10 μ M2.1 μ l each, probe of 10 μ M0.6 μ l, template RNA 2 μ l and DEPC water 11.2 μ l, mixing by vortex, centrifuging for a short time, adding the above mixture into a reaction tube of RPA lyophilized enzyme spheres, and adding magnesium acetate solution of 280mM 2.5 μ l;
according to the technical scheme, the invention provides a group of primers and probe reagents suitable for RPA, the reagents can accurately detect rabies viruses, have high specificity, sensitivity and accuracy, and make up for the defect that the conventional RPA detection technology lacks detection reagents with excellent effects.
Drawings
FIG. 1 shows the real-time fluorescence quantification of different nucleic acid samples amplified using the primers and probes of the present invention; wherein, curve A represents the nucleic acid RNA of rabies virus CVS-11 vaccine strain, and curve B represents the nucleic acid RNA of BD06 wild strain; C-G are other pathogenic nucleic acids: canine distemper virus nucleic acid, canine coronavirus nucleic acid, canine rotavirus nucleic acid and canine parvovirus nucleic acid, and healthy canine pharyngeal swab RNA;
FIG. 2 shows the real-time fluorescence quantification of wild type strain BD06 nucleic acid RNA at different copy numbers; wherein, the copy number represented by the curve from top to bottom is 105Copy/. mu.l, 104Copy/. mu.l, 103Copies/. mu.l and 102Copies/. mu.l.
Detailed Description
The invention discloses a reagent for detecting rabies virus and application thereof, and a person skilled in the art can appropriately improve process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the reagents and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations or modifications, as appropriate, may be made in the reagents and applications described herein to practice and use the techniques of this invention without departing from the spirit, scope, and spirit of the invention.
The reagent for detecting rabies viruses and the application thereof provided by the invention are further explained below.
Example 1: the reagent of the present invention
RV RPA FP (upstream primer): CTCTGGTGGAGATAAAACGTACTGATGTAG (SEQ ID NO: 1);
RV RPA RP (downstream primer): CTCAACCTATACAGACTCAAGAGAAGACCG (SEQ ID NO: 2);
RV RPA P (probe): AGGCATGGAACTGACAAGAGACCCCACTG (BHQ1-dT) C (THF) C (FAM-dT) GAGCATGCGTCCTTA (SEQ ID NO: 2);
example 2: the reagent of the present invention is used for specificity and sensitivity test
Materials: rabies virus CVS-11 strain, JX-08-45 strain and BD06 strain, canine distemper virus nucleic acid, canine coronavirus nucleic acid, canine rotavirus nucleic acid and canine parvovirus nucleic acid, and healthy canine pharyngeal swab. Example 1 primers and probes were synthesized by Shanghai Biotechnology Ltd, and all other reagents were biologically pure.
Nucleic acid extraction: RNA was extracted from each material using 200. mu.l of the culture or tissue homogenate obtained after centrifugation at 2000g according to the product instructions using the RNeasy Mini kit from QIAGEN.
In vitro transcription preparation of rabies virus template extraction of rabies virus BD06 strain RNA: performing RT-PCR amplification by using primers GACCATCRGCCCAATCAATTAAAAAGGG and CTGAGTCRGTGATGCTATGGTACC, purifying a PCR product, connecting the PCR product to a pGEM-T vector, identifying the direction of a recombinant plasmid, extracting the plasmid, performing restriction enzyme digestion to form linearity, performing In vitro Transcription according to the instruction of an In vitro Transcription T7Kit (Takara), digesting the product by DNaseI, precipitating the product by using 3M sodium acetate ethanol, dissolving the product In water without RNase, measuring the concentration, calculating the corresponding copy number, and sequentially performing 10-105Copy/. mu.l was diluted 10-fold to 100Individual copies/. mu.l were stored at-70 ℃.
RPA amplification Using the TwistAmp exo RT (TwistDx, Cambridge, UK) kit in 50. mu.l reaction system for RPA test, first add rehydration buffer 29.5. mu.l, 10. mu.M each 2.1. mu.l, 10. mu.M RV RPA P probe 0.6. mu.l, template RNA 2. mu.l and DEPC water 11.2. mu.l into 1.5ml centrifuge tube, vortex mixing, centrifuging briefly, add the above mixture into the reaction tube of RPA lyophilized enzyme ball, add 280mM magnesium acetate solution 2.5. mu.l, mix, place the reaction tube into RPA amplification detector or fluorescence quantitative PCR instrument, react at 39 ℃ for 25 minutes. The specific detection is carried out by taking different types of pathogenic nucleic acids and healthy dog total nucleic acids as templates, and the sensitivity detection of reaction is carried out by taking RNA standards of different dilutions as templates.
The primers and the probes designed by the invention are used for rapid detection, obvious amplification curves can be obtained by taking the RNA of rabies virus CVS-11 strain and BD06 strain as templates, and no amplification curve exists when other pathogenic nucleic acids such as canine distemper virus nucleic acid, canine coronavirus nucleic acid, canine rotavirus nucleic acid, canine parvovirus nucleic acid and total nucleic acid of healthy canine pharyngeal swab are taken as templates for RPA reaction (figure 1).
Viral nucleic acid was diluted 10-fold to multiple gradients with detection limits determined as102Single copy, with higher sensitivity (fig. 2).
Example 3: the accuracy test of the reagent of the invention
Samples from 12 home performance validation test samples were tested using the RPA method established in example 2. The nucleic acid templates in the samples to be tested were extracted using the QIAGEN RNeasy Mini Kit from Qiagen, and then the samples were tested for the presence of rabies virus by the RPA method. And simultaneously, the 12 samples are subjected to parallel detection by referring to a common RT-PCR method recommended by the world animal health Organization (OIE). The results show that the detection results of the two detection methods are consistent. The results show that the results obtained by the primer group, the probe and the detection method thereof are consistent with the results of RT-PCR, and the reliability of the invention is proved.
Example 4: comparison with other primer probes
According to the universal design principle of the RPA primers, 9 primers (4 upstream primers and 5 downstream primers) and 1 probe are designed according to a rabies virus genome, and different primer combinations are tested by taking the nucleic acid of the strain BD06 of the rabies virus as a template. In 20 groups of test results, 16 pairs of primers can obtain an amplification curve, but the fluorescence signals are weaker generally, while one pair of primers (F4 and R4) of the invention not only has short takeoff time of amplification but also has strong fluorescence signals, and the effect is far better than that of other primers.
TABLE 1
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
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<120> reagent for detecting rabies virus and application thereof
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Claims (4)
1. A reagent for detecting rabies viruses is characterized by comprising
1, and the upstream primer of the nucleotide sequence shown in SEQ ID NO: CTCTGGTGGAGATAAAACGTACTGATGTAG,
Downstream primer of the nucleotide sequence shown in SEQ ID NO. 2: CTCAACCTATACAGACTCAAGAGAAGACCG,
3 (BHQ1-dT) C (THF) C (FAM-dT) GAGCATGCGTCCTTA;
wherein BHQ1-dT represents thymine deoxynucleotide carrying fluorescence quenching group BHQ1, THF represents tetrahydrofuran linker, FAM-dT represents thymine deoxynucleotide carrying fluorescein group FAM.
2. Use of the reagent of claim 1 in the preparation of a kit for the detection of rabies virus.
3. The use of claim 2, wherein the rabies viruses are the CVS-11 strain rabies virus, the JX-08-45 strain rabies virus and the BD06 strain rabies virus.
4. A kit for detecting rabies virus, comprising the reagent according to claim 1; further comprising: rabies virus RNA extraction reagent and RPA amplification reaction reagent;
the RPA amplification reaction reagent comprises:
rehydration buffer 29.5. mu.l, DEPC water, RPA lyophilized enzyme spheres, and magnesium acetate solution.
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| CN109295260A (en) * | 2018-11-09 | 2019-02-01 | 辽宁佰昊生物科技有限公司 | For detecting and/or assisting detection to cause primer sets, reagent and the kit and detection method of hand-foot-and-mouth disease poison EV71 |
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| CN101153344B (en) * | 2007-09-30 | 2010-09-01 | 中国人民解放军军事医学科学院军事兽医研究所 | Series RT-PCR detection method and detection reagent kit for rabies viruses |
| CN101413948A (en) * | 2008-11-13 | 2009-04-22 | 浙江大学 | Rabies virus NP-ELISA antibody detection reagent kit |
| CN103361445B (en) * | 2013-07-30 | 2015-08-19 | 广西壮族自治区兽医研究所 | For detecting LAMP primer and the test kit thereof of rabies virus |
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