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Semi-nested RT-PCR detection method of canine astrovirus

A technology of RT-PCR and detection method, which is applied in the field of half-set RT-PCR detection of canine astrovirus, can solve the problems of specificity and sensitivity limitations, achieve specificity and sensitivity improvement, shorten the detection cycle, Overcoming the effect of long detection cycle

Inactive Publication Date: 2014-12-03
SHANGHAI JIAO TONG UNIV
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Problems solved by technology

[0004] Early detection of astroviruses is based on electron microscope observation and enzyme-linked immunosorbent technology. In recent years, with the application of molecular biology techniques, RT-PCR method has become an important means of detection of astroviruses, but they are limited in specificity, sensitivity, etc. There are certain limitations in

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  • Semi-nested RT-PCR detection method of canine astrovirus
  • Semi-nested RT-PCR detection method of canine astrovirus
  • Semi-nested RT-PCR detection method of canine astrovirus

Examples

Experimental program
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Embodiment

[0027] Example: Detection of Astrovirus in Dog Feces

[0028] 1. Primer design

[0029] Primers were designed targeting the conserved fragment of the canine astrovirus gene, and the primer sequences were as follows:

[0030] Coat Primer: Upstream Primer P1 L5′-CAANTCACAACCCAAAACAAA-3′

[0031] Downstream primer P2 L5′-CATTCACTTGGTCAAACACAGTC-3′

[0032] Inner set of primers: upstream primer P1 L5'-CAANTCACAACCCAAAACAAA-3'

[0033] Downstream primer P3 R5'-TTTTNACNATCACTGCTANGA-3'

[0034] 2. Extraction of total virus RNA in dog feces samples

[0035] Take 3g of dog feces samples and suspend them in 500ul PBS solution, take 200μl of the supernatant of the feces suspension and filter through a filter with a pore size of 0.45μl to remove eukaryotic and bacterial-sized particles. Add 1mL Trizol to the supernatant of 200ul feces suspension, and let stand at room temperature for 10min to completely denature the nucleoprotein complex; add 200ul chloroform and shake vigorously fo...

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Abstract

The invention provides a semi-nested RT-PCR detection method of canine astrovirus. The method comprises the following steps: 1, designing a primer with the conservative fragment of canine astrovirus gene as a target; 2, processing a detection sample, and extracting total RNA; 3, reversely transcribing the total RNA into cDNA; and 4, detecting the canine astrovirus in the sample through the semi-nested RT-PCR method. The method has the characteristics of high sensitivity, good specificity, simplicity, fastness and the like, and is suitable for the pathogenic diagnosis and the epidemiological studies of the canine astrovirus.

Description

[0001] This application is a divisional application of the following applications: filing date: December 23, 2011; application number: 201110440713.6; invention title: "half-set RT-PCR detection method for canine astrovirus". technical field [0002] The invention relates to biotechnology, in particular to a half-set RT-PCR detection method for canine astrovirus. Background technique [0003] Astroviruses are non-enveloped, single-stranded positive-sense RNA viruses with a five- or six-pointed star shape and belong to the family Astroviridae. Astrovirus was first discovered in 1975 by Madeley and Cosgrove in the stool of children with gastroenteritis detected by electron microscopy. Canine Astrovirus was first discovered in 1980 by Williams when he observed diarrheal dog feces with an electron microscope. Astroviruses can cause symptoms such as vomiting, diarrhea, and fever in humans, dogs, cats, and water leopards, especially in infants, young animals, the elderly, and tho...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/70C12Q1/6848
Inventor 崔立王春艳刘雨潇
Owner SHANGHAI JIAO TONG UNIV
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