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Triple PCR detection method for simultaneously detecting three feline diarrhea viruses and application of triple PCR detection method

A detection method and technology of diarrhea virus, which is applied in the field of triple PCR detection for simultaneous detection of three feline diarrhea viruses, can solve the problems of simultaneous detection and lack of efficient methods for clinical detection of enteroviruses, and achieve good stability and detection high efficiency effect

Pending Publication Date: 2021-10-22
SOUTHWEST UNIVERSITY FOR NATIONALITIES
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, the molecular detection method for FPV is very mature, but there is still a lack of efficient methods for the clinical detection of other enteroviruses. At present, there is no molecular detection method for simultaneous detection of FPV / FAstV / FECoV in China

Method used

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  • Triple PCR detection method for simultaneously detecting three feline diarrhea viruses and application of triple PCR detection method
  • Triple PCR detection method for simultaneously detecting three feline diarrhea viruses and application of triple PCR detection method
  • Triple PCR detection method for simultaneously detecting three feline diarrhea viruses and application of triple PCR detection method

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Experimental program
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Effect test

Embodiment 1

[0051] The primer sequences of the three feline diarrhea viruses designed in the present invention were synthesized by Shanghai Sangon Bioengineering Co., Ltd., and the primer information is shown in Table 1.

[0052] Table 1 Primer information

[0053]

Embodiment 2 3

[0054] The establishment of the triple PCR detection method of embodiment 2 three kinds of cat viruses

[0055] 1. Preparation of plasmid standards and extraction of plasmid DNA

[0056] Take FAstV, FPV and FECoV positive samples, extract nucleic acid according to the instructions of the DNA or RNA extraction kit or reverse transcribe into cDNA, and then amplify the target gene fragments respectively. The positive PCR products were purified, recovered and connected to the pMD19-T vector to construct three recombinant plasmids pMD19-T-FAstV ORF1b, pMD19-T-FPV VP2 and pMD19-T-FECoV N, which were respectively transformed into E. coli DH5α competent cells , Positive clones were screened out and sent to Sangon Bioengineering (Shanghai) Co., Ltd. for sequencing identification.

[0057] The positive bacteria with correct sequencing were amplified by shaking, and the plasmid DNA was extracted according to the instructions of the E.Z.N.A.TM Plasmid Kit plasmid extraction kit, and the ...

Embodiment 3 3

[0070] The inspection of embodiment 3 triple PCR method

[0071] (1) Specificity test

[0072] The mixed template of FAstV / FPV / FECoV, the single template of FAstV, FPV, FECoV and the nucleic acid of FNoV, FRV, FCV, CPV and CCoV were detected by the optimized triple PCR method to identify the specificity of the method.

[0073] From image 3 As a result, it can be seen that except for FPV, FAstV and FECoV, other pathogens cannot amplify the corresponding bands, indicating that the multiplex PCR method established by the method of the present invention has good specificity.

[0074] (2) Sensitivity test

[0075] The obtained three kinds of plasmid DNA templates were diluted 10-fold, and there were 8 gradients in total, each of which was 1×10 0 , 1×10 1 , 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 ; Take 0.5 μL DNA from each dilution gradient to mix well, and use the optimized triple PCR template to amplify to test the sensitivity of the method.

[0076] From ...

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Abstract

The invention discloses a triple PCR detection method for simultaneously detecting three feline diarrhea viruses and application of the triple PCR detection method. Specific primers for detecting cat astrovirus, cat parvovirus and cat intestinal coronavirus are provided, and the triple PCR detection method for the cat astrovirus, the cat parvovirus and the cat intestinal coronavirus is established. The specific primers and the detection method are high in specificity, high in sensitivity and good in repeatability and stability, and a rapid, sensitive and specific clinical detection method is provided for differential diagnosis of cat diarrheal diseases.

Description

technical field [0001] The invention belongs to the field of viral nucleic acid detection, and in particular relates to a triple PCR detection method for simultaneously detecting three feline diarrhea viruses and an application thereof. Background technique [0002] Diarrheal diseases in cats occur frequently clinically, among which the clinical symptoms caused by infectious pathogens are the most serious, especially the enterovirus represented by feline panleukopenia virus (Feline parvovirus, FPV) is the main cause of morbidity and death in young cats reason. In addition to FPV, viral metagenomics studies have shown that feline astrovirus (FAstV), feline enteric coronavirus (FECoV) and other mixed infections of enteroviruses also exist in cat feces samples , which may be an influencing factor in determining the efficacy and prognosis of clinical antiviral therapy. [0003] Feline parvovirus (Feline parvovirus, FPV) is also known as feline panleukopenia virus, feline diste...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686C12Q2600/16C12Q2537/143Y02A50/30
Inventor 黄坚李妍杨晓农郭琪
Owner SOUTHWEST UNIVERSITY FOR NATIONALITIES
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