CN113046489B - Multiplex RT-PCR primer set, kit and application for detecting porcine astrovirus - Google Patents

Abstract

The invention discloses a multiplex RT-PCR primer group for detecting porcine astrovirus, which comprises five pairs of specific primers, namely primers PAStV1-F and PAStV1-R, primers PAStV2-F and PAStV2-R, primers PAStV3-F and PAStV3-R, primers PAStV4-F and PAStV4-R and primers PAStV5-F and PAStV5-R, wherein the primers respectively have base sequences of sequence tables SEQ ID No.1 to SEQ ID No. 10. Accordingly, the inventor also prepares a corresponding RT-PCR kit and establishes a corresponding RT-PCR detection method. The method is simple and convenient in actual operation, reduces the operation times, greatly avoids cross contamination, and saves the detection time and the reagent consumption. The established detection method aims at the gene amplification of the virus conserved region, and has strong pertinence and high sensitivity. Can meet the requirements of simple, convenient, economic, convenient and accurate inspection in clinical experiments.

Description

Multiplex RT-PCR primer set, kit and application for detecting porcine astrovirus

technical field

The invention belongs to the field of biotechnology, and in particular relates to a multiple RT-PCR primer set, a kit and an application thereof for detecting porcine astrovirus.

Background technique

Porcine astrovirus was first discovered in pig feces by electron microscopy in 1980. Astroviruses are associated with enteric diarrheal disease in a wide variety of animals, from birds to mammals to humans. Porcine astrovirus often infects young, old and immunocompromised animals. At the same time, co-infections with rotavirus, coronavirus, calicivirus and other enteroviruses were often detected in porcine astrovirus-positive samples. There are many genotypes of porcine astrovirus, and 5 genotypes (PAstV1-PAstV5) have been found so far. The sequence homology of each genotype is not high, indicating that they come from different evolutionary branches. Although the genetic background of each genotype is significantly different, porcine astroviruses of different genotypes can be detected in the same area or even in the same pig, and mixed infection of PAstVs is common. In addition, co-infection of different genotypes of PAstV often leads to gene recombination, which has the potential risk of interspecies transmission and even zoonosis. Mixed infection of multiple genotypes often occurs between the genomes of PAstV, and two or more genotypes are infected, which further accelerates the genetic variation of the virus and brings challenges to the monitoring of porcine astrovirus. It is worth mentioning that the interspecies barrier of porcine astrovirus may not be strict, and the results of genetic evolution analysis suggest that porcine astrovirus may cross the interspecies barrier of humans and other animals. The researchers found that part of the ORF2 gene of the human Astrovirus and the porcine Astrovirus recombined, indicating that the porcine Astrovirus was closely related to the human Astrovirus during the evolution process. Therefore, the establishment of an efficient and fast detection method can monitor the infection of porcine astrovirus in pigs, analyze the recombination and mutation rules between strains, which will have a great impact on the biological characteristics, pathogenic mechanism and prevention of human and animal diseases of astrovirus. Co-infection with astroviruses is of great public health significance.

In today's intensively managed production, polygenotype mixed infection of porcine astrovirus generally occurs, and its epidemiological monitoring is a long-term and very necessary work. Establishing a fast and accurate The diagnostic approach becomes a powerful tool for the monitoring of PAstVs. In contrast, the establishment of multiple PCR detection technology can amplify multiple gene fragments at the same time, and the cost is low, fast, and cheap. It is undoubtedly the best detection method at present.

Contents of the invention

In order to solve the problems of cumbersome single RT-PCR operation and high risk of cross-contamination, the present invention provides a multiplex assay for porcine astrovirus detection that is easy to operate, avoids cross-contamination, saves detection time, reagent consumption, strong pertinence and high sensitivity. RT-PCR primer set, kit and its application, the specific scheme is as follows:

A multiplex RT-PCR primer set for detecting porcine astrovirus, including five pairs of specific primers, namely primers PAstV1-F and PAstV1-R, primers PAstV2-F and PAstV2-R, primers PAstV3-F and PAstV3-R , primers PAstV4-F and PAstV4-R, primers PAstV5-F and PAstV5-R, which respectively have the base sequences of SEQ.ID.No.1 to SEQ.ID.No.10 in the sequence table.

A multiplex RT-PCR kit for detecting porcine astrovirus, said kit comprising multiplex RT-PCR primer sets.

The kit also includes 5×buffer, dNTPMix, M-MLVReverseTranscriptae, RNase inhibitor, RNA, TarakaEx×Taq, 10×ExTaqBuffer, dNTPMix, TemplatecDNA and ddH ₂ O.

The application of the multiple RT-PCR primer set for detecting porcine astrovirus in RT-PCR amplification.

The RT-PCR amplification reaction system consists of 0.8 μmol/L of multiple RT-PCR primer sets, 0.25 μL of TarakaEx×Taq, 5 μL of 10×ExTaqBuffer, 4 μL of dNTPMix, 1 μL of each cDNA template, and filled to 50 μL with ddH ₂ O after sterilization.

The RT-PCR amplification reaction conditions are denaturation at 98°C for 10s, annealing at 55°C for 30s, extension at 72°C for 1min, and 30 cycles.

Advantages of the invention

1. In view of the current lack of effective and reliable technology for simultaneous detection and diagnosis of multi-genotype porcine astroviruses, the inventor has studied and designed five pairs of specific primers, which are respectively primers PAstV1-F and PAstV1-R, and primers PAstV2- F and PAstV2-R, primers PAstV3-F and PAstV3-R, primers PAstV4-F and PAstV4-R, primers PAstV5-F and PAstV5-R, and established the corresponding RT-PCR kit and established the corresponding RT -PCR detection method. The RT-PCR detection method can use the cDNA obtained from the total RNA of the same test sample as a template to carry out PCR amplification, and according to the size of the amplified product, the genotype of the virus infected by the sample to be tested can be judged by agarose gel electrophoresis to realize Simultaneously amplifying five genotypes of the virus in the same reaction system for identification has an important practical effect on early detection and effective prevention and control of the occurrence and spread of porcine astrovirus.

2. The present invention is simple in actual operation, reduces the number of operations, greatly avoids cross-contamination, and saves detection time and reagent consumption. Since the established detection method is aimed at the gene amplification of the conserved region of the virus, it has strong pertinence and high sensitivity. In clinical trials, the test requirements of simplicity, economy, convenience and accuracy can be met. Provided technical support for understanding the prevalence of porcine astrovirus, disease prevention and control. It laid a foundation for monitoring the prevalence of porcine astrovirus types 1-5, mixed infection and molecular biology rapid diagnosis.

Description of drawings

Figure 1 is the specific primer detection diagram corresponding to PAstV1, PAstV2, PAstV3, PAstV4, and PAstV5. The lanes in the figure are M:M:DL2000 DNA Marker from left to right; A, B, C, D, and E are PAstV1, PAstV2, PAstV3, PAstV4, PAstV5;

Figure 2 is a multiplex RT-PCR primer concentration optimization diagram, where M: DL2000 DNA Marker; 1, 2, 3, 4, 5 primer concentrations are 0.2μmol/L, 0.4μmol/L, 0.6μmol/L, 0.8μmol /L, 1.0 μmol/L.

Figure 3 is an optimization diagram of the multiplex RT-PCR annealing temperature, where the annealing temperatures of M:DL2000 DNA Marker; 1, 2, 3, 4, and 5 are 53°C; 54°C; 55°C; 56°C; 57°C.

Figure 4 is a diagram of the primer-specific detection results corresponding to the simultaneous detection of PAstV1, PAstV2, PAstV3, PAstV4, and PAstV5 in the present invention. The lanes in the figure are from left to right: M: DL2000 DNA Marker; 1: PAstV1; 2: PAstV2; 3 : PAstV3; 4: PAstV4; 5: PAstV5; 6: PAstV1, PAstV2, PAstV3, PAstV4 and PAstV5; 7-13 non-target genes; 14: negative control.

Fig. 5 is the result figure of the single sensitivity experiment of simultaneous detection of PAstV1, PAstV2, PAstV3, PAstV4 and PAstV5 in the present invention, and the swimming lanes are as follows from left to right: 1:10ng; 2:1ng; 3:100pg; 4:10pg; 5:1pg ; 6: 0.1 pg; 7: negative control positive plasmid.

Figure 6 is a diagram of the detection results of simultaneous detection of multiple RT-PCR sensitivity. The lanes in the figure are from left to right: 1:10ng; 2:1ng; 3:100pg; 4:10pg; 5:1pg; 6:0.1pg 7: Negative control positive plasmid.

positive plasmid.

detailed description

The present invention will be further explained and illustrated below in conjunction with the accompanying drawings and specific examples, and the following specific examples do not limit the protection scope of the present invention. Unless otherwise specified, the technical means in the embodiments are conventional means well known to those skilled in the art.

Example 1

1.1 Test strains and clinical samples

Porcine Astrovirus Type 1, Porcine Astrovirus Type 2, Porcine Astrovirus Type 3, Porcine Astrovirus Type 4, Porcine Astrovirus Type 5, Porcine Enterovirus, Porcine Seneca Virus, Porcine Pseudorabies Virus , classical swine fever virus, and porcine reproductive and respiratory syndrome virus positive nucleic acid samples were all identified by our laboratory and stored at -80°C. Porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, porcine rotavirus triple live vaccine (Harbin Veken Biotechnology Development Company), porcine Japanese encephalitis virus (Wuhan Keqian Biotechnology Co., Ltd.). The clinical stool samples were all from a pig farm in Guangxi in 2020, with a total of 275 samples.

1.2 Primer design and synthesis

According to the whole genome sequences of PAstV1, PAstV2, PAstV3, PAstV4 and PAstV5 reported on GenBank, the genomes of different strains were compared and analyzed, and their relatively conservative nucleic acid fragments were screened. Five pairs of primers targeting the above-mentioned Specific primers for five viruses, and online analysis of five pairs of primers by NCBI. Finally, five pairs of primers (Table 1) were screened out, and the primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. Five pairs of primers can specifically amplify specific fragments of 125 (PAstV1), 573 (PAstV2), 175 (PAstV3), 485 (PAstV4), and 305 (PAstV5).

Table 1 Multiplex RT-PCR Primer Sequence List

Embodiment 2 cDNA template preparation

2.1 Extraction of RNA from samples to be tested: collect pig feces, dilute the feces samples by vortexing with PBS, centrifuge at 8000r/min at 4°C, take the supernatant, and store at -80°C.

2.2 Reaction conditions for reverse transcription: react at 42°C for 1 hour, and store the obtained cDNA at -20°C for PCR amplification.

2.3 Table 2 Reverse transcription PCR system

2.4 cDNA extraction: extract the DNA of PAstV1, PAstV2, PAstV3, PAstV4, and PAstV5 according to conventional methods, and store at -20°C.

Embodiment 3 Single PCR amplification sequence verification

The cDNAs of PAstV1, PAstV2, PAstV3, PAstV4, and PAstV5 preserved in Example 2 were used as templates, and the five pairs of specific primers designed in Example 1 were used for single PCR amplification. The single PCR amplification system is shown in Table 3. The reaction conditions were denaturation at 98°C for 10s; annealing at 55°C for 30s; extension at 72°C for 1min; 30 cycles. RT-PCR products were detected by 1.5% agarose gel electrophoresis. The test results are shown in Figure 1. The above PCR products were purified with a purification kit for the target gene, and sequenced by Sangon Bioengineering (Shanghai) Co., Ltd. Sequencing results showed that it was consistent with the nucleotide sequence of the corresponding strain.

Table 3 RT-PCR reaction system

Reagent 25μL reaction system Taraka Ex×Taq 0.25 μL 10×Ex Taq Buffer 5μL dNTP Mix 4μL upstream primer 0.8μmol/L downstream primer 0.8μmol/L Template cDNA 1μL each ddH₂O Make up to 50μL

Embodiment 4 multiplex RT-PCR reaction

4.1 Multiplex RT-PCR reaction system and optimization conditions;

Under the same reaction conditions (denaturation at 98°C for 10 s; annealing at 55°C for 30 s; extension at 72°C for 1 min; 30 cycles), PAstV1, PAstV2, PAstV3, PAstV4, and PAstV5 at the same concentration were mixed as multiplex RT-PCR templates , optimize multiple RT-PCR reaction conditions, including primer concentration and reaction system.

Primer concentration screening: Reaction system used: upstream and downstream primers 1.0 μL each; TarakaEx×Taq 0.25 μL; 10×ExTaqBuffer 5 μL; dNTPMix ₄ μL; cDNA template 2.5 μL; A total of 5 different concentration gradients of 0.4 μmol/L, 0.6 μmol/L, 0.8 μmol/L, and 1.0 μmol/L were used for PCR amplification, and primer concentration screening was performed. The results are shown in Figure 2.

Annealing stability optimization: the annealing temperature was selected as 53°C; 54°C; 55°C; 56°C; 5:57°C with 5 different gradients for PCR amplification, and the optimal annealing temperature was selected. The results are shown in Figure 3.

4.2 Multiplex RT-PCR specificity;

The mixed cDNA templates of PAstV1, PAstV2, PAstV3, PAstV4 and PAstV5 and the cDNA of positive samples of PEV, PRRSV, SVV, CSFV, JEV and porcine triple live vaccine (PEDV, RV, TGEV) were established under the conditions of multiple RT-PCR reactions. The DNA template of the PRV-positive sample was amplified, and the amplification results showed (as shown in Figure 4) that only PAstV1, PAstV2, PAstV3, PAstV4 and PAstV5 single cDNA and PAstV1, PAstV2, PAstV3, PAstV4 and PAstV5 mixed cDNA amplified and the target fragment The size fits the band.

4.3 Single/multiple RT-PCR sensitivity test

The cDNA concentration of PAstV1, PAstV2, PAstV3, PAstV4 and PAstV5 was measured by spectrophotometer and adjusted to 1:10ng; 2:1ng; 3:100pg; 4:10pg, 5:0.1pg; 6:1pg; 7:0.1pg The cDNA of each concentration was used as a template for PCR amplification, and the minimum detection amount was detected.

As shown in Figure 5, the 5 virus genotype plasmids are subjected to sensitivity amplification, and the minimum detection amount of cDNA of PAstV1, PAstV2, PAstV3, PAstV4 and PAstV5 by this method is 0.1pg, 0.1pg, 1pg, 1pg, 0.1pg respectively. Fig. 6, 5 kinds of viral genotype plasmids are mixed for sensitive amplification, and the minimum detectable amount of cDNA of PAstV1, PAstV2, PAstV3, PAstV4 and PAstV5 by this method is 10pg.

Example 5 Preliminary Application of Clinical Samples

Use the multiplex RT-PCR primers of the present invention to perform RT-PCR detection of the following samples, and detect 275 feces collected from various pig farms in Guangxi in 2020.

Table 4 Clinical Sample Test Results

As an enteropathogenic virus, astrovirus has been circulating in many countries around the world since it was first discovered. For quite a period of time, its clinical symptoms were considered to be sporadic mild diarrhea. The pathogenicity of astrovirus is not Did not attract attention. With the development of detection methods, we have a deeper understanding of astroviruses. Astrovirus infection is also accompanied by nephritis, hepatitis, and neurological symptoms. Other studies have pointed out that the astrovirus has cross-species gene recombination.

Pork is one of the main meats in our country. With the rapid development of intensification, the price of pork continues to rise. The aquaculture industry is characterized by a wide distribution range and a large scale of aquaculture. The large-scale aquaculture method makes the infectivity of astrovirus remain high. Although the cause of porcine diarrhea virus is not only astrovirus, but astrovirus is often mixed with other diarrhea viruses, and often acts as an "accomplice" in diarrhea viruses. Moreover, there is a mixed infection of multiple genotypes of astrovirus, which aggravates the symptoms of diarrhea, or causes symptoms of nerve or encephalitis, often causing economic losses to farmers. At present, most of the diagnostic tests for astrovirus stay at the detection level of single-plex RT-PCR or double RT-PCR, while other detection methods such as direct immunofluorescence test, electron microscopy, agar immunodiffusion test are expensive and not Right-to-scale clinical testing. Therefore, establishing a multiplex RT-PCR detection method for different genotypes is efficient and cost-effective.

The invention aims at distinguishing the mixed infection situation of each genotype of the porcine astrovirus, and establishes a multiple RT-PCR detection kit. The detected objects involve AstV1, PAstV2, PAstV3, PAstV4 and PAstV5. Some genotypes of the above-mentioned genes are more harmful in pig herds, and their potential public health significance is particularly important, and they are of great value in detecting the prevalence and prevention of epidemic diseases.

The multiple RT-PCR established by the invention has high sensitivity, and the minimum detection amount reaches 10 pg; and it can specifically amplify the target gene; the requirement for the extracted RNA content is low, and the reaction system is simple and easy to operate.

In summary, the present invention provides a specific and sensitive detection and identification of each genotype of PAstV1, PAstV2, PAstV3, PAstV4 and PAstV5 and the mixed infection situation, which has important application value in public epidemiological investigations, etc. .

The above-described embodiments are preferred embodiments of the present invention, and are only used to explain the present invention, not to limit the scope of the present invention. For those skilled in the art, they can pass the It is easy to make other implementations by means of replacement or change, so all changes and improvements made on the principle of the present invention should be included in the patent scope of the present invention.

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Claims (6)

1. The multiplex RT-PCR primer group for detecting the porcine astrovirus is characterized by comprising five pairs of specific primers, namely primers PAStV1-F and PAStV1-R, primers PAStV2-F and PAStV2-R, primers PAStV3-F and PAStV3-R, primers PAStV4-F and PAStV4-R and primers PAStV5-F and PAStV5-R, wherein nucleotide sequences of the primers are respectively base sequences shown in a sequence table SEQ ID No.1 to SEQ ID No. 10.

2. A multiplex RT-PCR kit for detecting porcine astrovirus, the kit comprising the multiplex RT-PCR primer set of claim 1.

3. The multiplex RT-PCR kit for detecting porcine astrovirus according to claim 2, wherein the kit further comprises 5 xbuffer, dNTPmix, M-MLV Reverse transcriptase, RNase inhibitor, RNA, taraka Ex xTaq, 10 xExTaq Buffer, dNTPmix, template cDNA and ddH ₂ O。

4. Use of the multiplex RT-PCR primer set according to claim 1 for RT-PCR amplification of porcine astrovirus for non-diagnostic purposes.

5. The use of claim 4, wherein the RT-PCR amplification reaction system comprises multiple RT-PCR primer sets of 0.8 μmol/L each, 0.25 μ L of Taraka Ex xTaq, 5 μ L of 10 xEx Taq Buffer, 1 μ L of dNTP Mix4 μ L, cDNA template each, and sterilized ddH ₂ The content of O is filled to 50 mu L.

6. The use of claim 4, wherein the RT-PCR amplification reaction conditions are 98 ^o C modified 10s,55 ^o C anneal 30s,72 ^o C extension is 1min,30 cycles.

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