CN111334617A - Multiplex PCR detection primer for detecting four viruses of dog, detection method and application - Google Patents

Abstract

The invention discloses a multiplex PCR primer for detecting four viruses of a dog, a detection method and application, wherein the primer comprises the following components: primers CCV-F and CCV-R for detecting canine coronavirus; primers CPIV-F and CPIV-R for detecting canine parainfluenza virus; and primers CAV-F and CAV-R for detecting canine adenovirus type I and canine adenovirus type II. The PCR detection method established by the primer can detect single virus, can simultaneously detect mixed infection of four viruses of CCV, CPIV, CAV-1 and CAV-2, has the advantages of high detection sensitivity, good specificity and the like, can reduce the occurrence of false positive, can realize the rapid, accurate, sensitive and early detection of pathogen targets, provides technical support for establishing a standardized detection method for dog health monitoring, and can greatly save time and cost.

Description

Multiplex PCR detection primer for detecting four viruses of dog, detection method and application

Technical Field

The invention relates to the technical field of biological detection, in particular to a multiplex PCR detection primer for detecting four viruses of a dog, a detection method and application.

Background

Canine Coronavirus (CCV), Canine parainfluenza virus (CPIV), Canine infectious hepatitis virus (ICHV), and Canine infectious laryngotracheitis virus (ICLV) are major pathogens that cause Canine diseases. CCV can cause death in sick dogs, particularly in sick puppies; canine parainfluenza belongs to one of common diseases of dogs, and after the dogs are infected with CPIV, a series of secondary infections are often caused, and the dogs can die when being serious; canine infectious hepatitis is a highly contact, acute and septic infectious disease, and the etiology is Canine adenovirus type I (CAV-1). Once the disease occurs, the death rate of puppies under the age of 1 year is high, and the disease is clinically often mixed with canine distemper to cause infection, so that the disease is extremely harmful; the Canine infectious laryngotracheitis is caused by Canine adenovirus type II (CAV-2), is a Canine susceptible chronic respiratory infectious disease, and often causes symptoms such as laryngitis, tracheitis, bronchitis and the like.

Diseases caused by canine parainfluenza virus and canine infectious laryngotracheitis virus belong to canine respiratory diseases, and are common diseases, which occur all the year round, but have less morbidity in summer as a whole, and have higher morbidity and mortality in other seasons and season changes. The dog respiratory diseases can be caused by various factors, such as viruses, bacteria, fungi, parasites and the like, and in addition, the dog respiratory diseases can be caused by pollen, allergy caused by medicaments, foreign body inhalation and other physical factors. The common clinical symptoms of canine respiratory diseases are similar, mainly: elevated body temperature, coughing, sneezing, nasal discharge, etc., which typically self-heal in a short period of time, and are non-lethal unless other factors are involved, such as mixed infections. Therefore, the diagnosis is difficult only from the aspect of visual diagnosis, further detection is needed by means of an X-ray machine, a blood routine and the like, and the canine respiratory diseases are often infected by two or more than two kinds of mixed infection, so that the diagnosis of the diseases becomes more difficult. Because the diseases are often two or more mixed infections, the clinical symptoms are similar, the treatment period is long, and the like, and the diagnosis difficulty is high due to a plurality of factors, the judgment of the correct etiology in the aspect of clinical treatment is highly valued by relevant personnel in the canine raising industry, veterinary doctors and the like.

The canine infectious hepatitis virus and the canine infectious laryngotracheitis virus are respectively infected by canine adenovirus type I and type II, the canine adenovirus is a virus with the strongest pathogenicity in the mammalian adenovirus genus currently known, the genome of the canine adenovirus virus contains 4 early transcription regions which are respectively E1, E2, E3 and E4, the main difference of the gene sequences of the canine infectious hepatitis virus and the canine infectious laryngotracheitis virus is in an E3 region, and CAV-2 is more than CAV-1 by about 500bp in an E3 region. The pathogenicity of the canine infectious hepatitis virus and the canine infectious laryngotracheitis virus is strong, but the canine infectious hepatitis virus is easy to infect puppies under the age of 1 year, has high lethality rate, is frequently mixed with canine distemper to infect clinically, and can be poisoned for a long time even after recovery, so that although the cases found by the canine infectious hepatitis virus are few, the canine infectious hepatitis virus has huge harm to various industries of domestic dog breeding. CAV-2 is a chronic respiratory infectious disease. CAV-2 immunity can effectively aim at CAV-1, CAV infection in China is quite common, and dogs of any age, sex and breed are susceptible, so that CAV-2 research significance is great.

The canine coronavirus, the canine distemper virus and the canine parvovirus belong to common virulent infectious diseases of dogs, but compared with the former two viruses, the canine coronavirus is obviously inadequately attached to people in daily life, the canine coronavirus is mainly manifested by gastroenteritis, the clinical symptoms are vomiting and diarrhea, and the canine coronavirus has no characteristic indexes in the aspects of clinical symptoms, pathological anatomy, epidemiology and the like, so the canine coronavirus is difficult to detect, laboratory detection means such as electron microscope observation and the like are usually required for detection, and the canine coronavirus is recurred after clinical constant disappearance within a period of time, and has serious harm to the environment and the health of dogs.

The canine coronavirus, the canine infectious hepatitis virus, the canine infectious laryngotracheitis virus and the canine parainfluenza virus are four viruses which are susceptible to the canine, bring serious economic benefit loss to the canine breeding industry in China, and need to be monitored regularly in the breeding process. In recent years, various diagnostic methods have been established for each virus, but standardization has not been established. The common serological diagnosis method is easy to form missed detection when the virus content is low, and the colloidal gold method has high detection sensitivity but is easy to generate false positive results. In addition, because the four viruses often have the phenomenon of mixed infection, a method for simultaneously detecting the mixed infection of the four viruses is established, and the establishment of a standardized detection method for dog health monitoring is extremely important.

Disclosure of Invention

The invention aims to provide a multiplex PCR primer, a detection method and application for detecting four viruses of a dog, and three pairs of multiplex PCR detection primers are designed, and the PCR detection method established by the primers can simultaneously detect mixed infection of the four viruses of CCV, CPIV, CAV-1 and CAV-2.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides a multiplex PCR primer for detecting four viruses of a dog, which comprises the following components:

primers CCV-F and CCV-R for detecting canine coronavirus are respectively shown as SEQ ID NO: 1 and SEQ ID NO: 2 is shown in the specification;

primers CPIV-F and CPIV-R for detecting canine parainfluenza virus, shown in SEQ ID NO: 3 and SEQ ID NO: 4 is shown in the specification; and

primers CAV-F and CAV-R for detecting canine adenovirus type I and canine adenovirus type II are shown as SEQ ID NO: 5 and SEQ ID NO: and 6.

The invention also provides a multiplex PCR method for detecting four viruses of a dog by using the primers, which comprises a 50 mu L multiplex PCR reaction system, namely 0.5 mu L of each of 2 × easy Taq Super Mix 25 mu L, CCV-F and CCV-R, 0.5 mu L of each of canine adenovirus I type and canine adenovirus II type CAV-F and CAV-R, 0.5 mu L, CAV-F and CAV-R, 1.2 mu L of canine coronavirus template, 1.2 mu L of canine parainfluenza virus template, 1.2 mu L of canine adenovirus I type and canine adenovirus II type template, and 16.2 mu L of ddH2O 16.2;

preferably, the method further comprises the following steps: multiplex PCR reaction procedure: 94 ℃ for 5 min; 30s at 94 ℃; 30s at 55 ℃; 72 ℃, 90s, 30 cycles; 72 ℃ for 10 min; storing at 4 ℃.

The invention also provides application of the primer in a multiplex PCR detection kit for detecting four canine viruses.

The invention also provides application of the primer in preparation of a reagent for detecting single infection or mixed infection of four viruses of a dog.

The invention also provides application of the multiplex PCR method in a multiplex PCR detection kit for detecting four canine viruses.

Preferably, the reagent is applied to preparing reagents for detecting single infection or mixed infection of the four viruses of the dog.

The invention discloses the following technical effects:

the invention respectively carries out homology analysis on the genome sequences of CCV, CPIV, CAV-1 and CAV-2 viruses aiming at the whole genome sequences of the CCV, the CPIV and the CAV-1 and CAV-2 viruses, and respectively designs primers according to the sequence on the S gene of the CCV, the segment on the NP gene of the CPIV and the segment of the E3 gene of the CAV-1 and CAV-2 to form the multiplex PCR detection primers for detecting the four common viruses of the dog. The three pairs of primers are used for PCR detection of four suspected virus pathological materials clinically by adopting a mode of mixing equal concentrations and equal proportions, and the primers can detect not only single infection pathological materials but also mixed infection of four viruses, have the advantages of high sensitivity and good specificity, can reduce the probability of false positive, realize the aim of quickly, accurately, sensitively and early identifying pathogens, provide technical support for establishing a standardized detection method for health monitoring of dogs, and greatly save time and cost.

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 results of single PCR amplification of CCV, CPIV, CAV-1 and CAV-2 viruses of the present invention; a: m, Marker; first lane, CCV virus; b: m, Marker; first lane, CPIV virus; c: m, Marker; first lane, CAV-I virus; d: m, Marker; first lane, CAV-II virus;

FIG. 2 shows the results of multiplex PCR amplification of CCV, CPIV, CAV-1 and CAV-2 viruses of the present invention; m, Marker; first lane, four virus pools;

FIG. 3 shows the results of multiplex PCR susceptibility testing of CCV, CPIV, CAV-1 and CAV-2 viruses according to the present invention; m: DL2000DNA Marker; first lane: the template concentration is 400 ng/. mu.L; first lane 2: the template concentration is 200 ng/. mu.L; a third lane: the template concentration is 100 ng/. mu.L; a fourth lane: the template concentration was 50 ng/. mu.L.

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 specification and examples are exemplary 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

Design of multiplex PCR primers for detecting four canine viruses

1. Experimental Material

The strains CCV and CAV-2 are from the microbial and immune laboratory preservation of animal medical college of Qingdao agricultural university, and the CPIV and CAV-1 strains are bought from ATCC by Beinai Chuan union.

2. Experimental methods

The genome sequences of CCV, CPIV, CAV-1 and CAV-2 viruses were retrieved from GenBank databases, the genome sequences of the viruses were analyzed for homology, and the sequences of the conserved regions of the four viruses were determined by examining the literature, and primers were designed using primer design software such as primer 5.0 using the sequence of the S gene of CCV, the fragment of the NP gene of CPIV and the fragment of the E3 gene of CAV-1 and CAV-2 (as shown in Table 1).

TABLE 1 multiplex PCR primer sequences

3. Extraction of RNA and DNA

The method comprises the steps of extracting DNA of positive strains of canine infectious hepatitis virus and canine infectious laryngotracheitis virus by using a virus genome DNA/RNA extraction kit purchased from Tiangen Biochemical technology limited company as a template, extracting RNA of the positive strains of canine coronavirus and canine parainfluenza virus, carrying out reverse transcription by using a reverse transcription kit purchased from Taoist physician technology limited company, taking obtained cDNA as the template, adjusting the concentration of the templates of the four viruses, mixing, carrying out single PCR amplification by using primers of the four viruses respectively, and carrying out quadruple PCR amplification on the primers of the four viruses.

4. Establishment of multiplex PCR detection method

The PCR amplification conditions are shown in Table 2.

TABLE 2 multiplex PCR System

PCR reaction procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30 s; annealing at 55 ℃ for 30 s; extension at 72 ℃ for 90s for 30 cycles; final extension at 72 deg.C for 10 min; storing at 4 ℃.

5. Electrophoretic detection

Weighing 0.2g of agarose, placing the agarose in a conical flask, adding 20mL of 1 × TAE, sealing the agarose in a tin foil paper, placing the conical flask in a microwave oven for multiple times of boiling until the solution is clear, dropwise adding 3 mu L of nucleic acid dye when the temperature of the solution is reduced to 50-60 ℃, shaking and uniformly mixing, pouring the mixture into a mold, waiting for 15-20min, placing the mixture into an electrophoresis tank after gel forming, adding 1 × TAE into the electrophoresis tank until the solution just submerges the gel, respectively adding 10 mu L of LDL 2000DNA Marker and 10 mu L of PCR amplification product into a sample application hole, immediately electrifying after sample application, setting the voltage to be 120V, placing the electrophoresis result under a gel imager after running for 20-30min, opening ultraviolet, and observing the amplification length and brightness.

6. Results

When the multiplex PCR method is established, the annealing temperature is optimized, PCR amplification bands with the annealing temperature of 57 ℃ become fuzzy and have a dragging phenomenon, when the annealing temperature is 55 ℃, electrophoresis bands amplified by PCR are bright and clear, the effect difference is obvious, and the annealing temperature is used for multiplex PCR when the optimal annealing temperature is 55 ℃.

The lengths of the fragments obtained by the amplification of the CCV, CPIV, CAV-1 and CAV-2 primers are respectively 445bp, 577bp, 501bp and 1023bp, which are consistent with the reality, as shown in FIG. 1 and FIG. 2, FIG. 1 shows the single PCR of each virus, and FIG. 2 shows the multiple PCR result.

Example 2

Specificity test for multiplex PCR

The method comprises the steps of clinically collecting dog excrement anal swabs and nasal swabs, taking the obtained swabs as templates, extracting DNA/RNA from clinically collected samples by using a virus genome DNA/RNA extraction kit, carrying out PCR amplification after RNA is reversely transcribed into cDNA, selecting cDNA of canine coronavirus and canine parainfluenza virus positive morbid materials obtained by amplification, DNA of canine infectious hepatitis virus and canine infectious laryngotracheitis virus positive morbid materials, cDNA and DNA extracted from the anal swabs and the nasal swabs of a healthy dog, and taking the canine parvovirus positive morbid materials as templates, and carrying out multiple PCR in clinical specific tests. Adding CPIV and CAV primers into a CCV template; adding CCV and CAV primers into a CPIV template; adding primers of CCV and CPIV to the templates of CAV-1 and CAV-2; three primers, CCV, CPIV and CAV, were added to the extracted template of a healthy dog and the template of canine parvovirus, PCR amplification was performed using the reaction conditions of multiplex PCR in example 1, and identification was performed by agarose gel electrophoresis.

The results show that: DNA extracted from anal swab and nasal swab of healthy dog is used as template, extracted RNA is used as template after being reverse transcribed into cDNA, and cDNA extracted from canine parvovirus positive morbid material and reverse transcribed by using kit is used as template, and multiple PCR amplification is respectively carried out, after agarose gel electrophoresis, the amplified band is not generated when a gel imager is used for watching.

Example 3

Multiplex PCR sensitivity assay

The cDNA and DNA were adjusted to a final concentration of 400 ng/. mu.L using the cDNA of the canine coronavirus and canine parainfluenza virus positive strains, and the DNA of the canine infectious hepatitis virus and canine infectious laryngotracheitis virus positive strains as templates using a spectrophotometer, and then subjected to 4-fold 2-fold dilution, and divided into 4 gradients of 400 ng/. mu.L, 200 ng/. mu.L, 100 ng/. mu.L, and 50 ng/. mu.L, respectively, and PCR amplification was performed using the reaction conditions of multiplex PCR in example 1, and then identified by agarose gel electrophoresis.

The results show that: as shown in FIG. 3, clear bands were observed on the gel imager after 4 gradient concentrations were electrophoresed, but the template concentration began to decrease in clarity after being lower than 100 ng/. mu.L, and the PCR amplification results were less effective than those of higher concentrations.

Example 4

Clinical validation of multiplex PCR

The positive detection was carried out by randomly collecting 250 parts each of nasal swabs and anal swabs in Nanchang police dog base, 8 parts of CCV positive morbid material collected from Nanchang police dog base, 1 part of CPIV and 1 part of CAV-2 positive morbid material, and 32 parts total of nasal swabs and anal swabs of dogs with respiratory symptoms and diarrhea symptoms collected from other dog base. Multiplex PCR assay was as in example 1.

The results shown in table 3 show that: the multiple PCR method is used for finding that 15 parts of CCV, 3 parts of CPIV and 5 parts of CAV-2 are present, and no CAV-1 is detected, and the analysis reason is considered to be that the time for collecting a large amount of disease in summer is probably that canine infectious diseases are less, and in addition, CAV-1 generally infects puppies under the age of 1 year, the morbidity and mortality are high, and the number of live and toxic dogs is less. Detection with colloidal gold revealed 17 parts of CCV, 3 parts of CPIV and 5 parts of CAV-2, and no CAV-1 was detected. The results were as expected.

Table 3 clinical verification results

Example 5

Multiplex PCR repeatability detection

2 positive pathogens are selected for each virus detected in the example 4, 10 times of repeated detection is carried out, and the electrophoresis results of multiple PCR amplification are found to have no obvious difference, so that the multiplex PCR method can be obtained, and is reusable and high in stability.

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.

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Claims (7)

1. A multiplex PCR primer for detecting four canine viruses, which is characterized by comprising:

primers CCV-F and CCV-R for detecting canine coronavirus are respectively shown as SEQ ID NO: 1 and SEQ ID NO: 2 is shown in the specification;

primers CPIV-F and CPIV-R for detecting canine parainfluenza virus, shown in SEQ ID NO: 3 and SEQ ID NO: 4 is shown in the specification; and

primers CAV-F and CAV-R for detecting canine adenovirus type I and canine adenovirus type II are shown as SEQ ID NO: 5 and SEQ ID NO: and 6.

2. The multiplex PCR method for detecting four canine viruses using the primers as set forth in claim 1, which comprises a 50. mu.L multiplex PCR reaction system of 2 × easy Taq Super Mix 25. mu. L, CCV-F and CCV-R each 0.5. mu. L, CAV-F and CAV-R each 0.5. mu.L, canine adenovirus type I and canine adenovirus type II CAV-F and CAV-R each 0.5. mu.L, canine coronavirus template 1.2. mu.L, canine parainfluenza virus template 1.2. mu.L, canine adenovirus type I and canine adenovirus type II template each 1.2. mu.L and ddH₂O 16.2μL。

3. The multiplex PCR method for primer detection of four canine viruses according to claim 2, further comprising: multiplex PCR reaction procedure: 94 ℃ for 5 min; 30s at 94 ℃; 30s at 55 ℃; 72 ℃, 90s, 30 cycles; 72 ℃ for 10 min; storing at 4 ℃.

4. Use of the primers of claim 1 in a multiplex PCR assay kit for detecting four canine viruses.

5. Use of the primer of claim 1 in the preparation of a reagent for detecting single infection or mixed infection of four viruses in a dog.

6. Use of the multiplex PCR method according to any one of claims 2 to 3 in a multiplex PCR detection kit for detecting four canine viruses.

7. The use of claim 6 for the preparation of a reagent for the detection of canine single infection or mixed infection with four viruses.

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