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Multiple PCR detection primer group and kit for rapidly differentiating PCV1 type from PCV3 type

A detection kit, PCV1 technology, applied in the field of diagnosis in the field of veterinary biotechnology, can solve the problem that porcine circovirus is difficult to meet the actual needs, and achieve the effect of strong sensitivity, good repeatability and strong specificity

Inactive Publication Date: 2018-02-02
INST OF ANIMAL HUSBANDRY & VETERINARY MEDICINE HENAN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, there are few studies on PCV3, and the original detection and typing methods for porcine circovirus have been difficult to meet the actual needs

Method used

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  • Multiple PCR detection primer group and kit for rapidly differentiating PCV1 type from PCV3 type
  • Multiple PCR detection primer group and kit for rapidly differentiating PCV1 type from PCV3 type
  • Multiple PCR detection primer group and kit for rapidly differentiating PCV1 type from PCV3 type

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Experimental program
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Effect test

Embodiment 1

[0032] The specific implementation of the present invention will be described in further detail below in conjunction with the examples. Unless otherwise specified, the experimental methods used in the following examples are conventional methods in the art, or follow the conditions and implementation steps suggested by the manufacturer. Embodiment 1, the construction of detection method

[0033] 1. Primer design

[0034] According to the PCV1 and PCV3 sequences included in GenBank, by comparing the differences between PCV1 and PCV3, design specific primers L1, L2 and L3, as follows:

[0035] L1: 5'-ACCAGCGCACTTCGGCAGCGGCAGCA-3' (SEQ ID NO.1)

[0036] L2: 5'-GCATTGTTCACCAGTACCCA-3' (SEQ ID NO.2)

[0037] L3: 5'-AAGTCCCCTTCCTGCGTCCGCTAATCT-3' (SEQ ID NO.3)

[0038] Using L1, L2 and L3 for PCR amplification, the estimated size of the amplified fragment for PCV1 type is 901bp, and the predicted size of the amplified fragment for PCV3 type is 152bp.

[0039] 2. Extraction of vi...

Embodiment 2

[0069] Embodiment 2, primer action verification

[0070]Taking PCV1 type as a sample, use primer design software to design primer L4 primer 5'-CGGTATATCCACTCACACG-3' (SEQ ID NO.4) according to the sequence, and use the method in Example 1 to detect with the above-mentioned L1 primer, and the annealing temperature is 53 °C, 54 °C, 55 °C, 56 °C, 57 °C, 58 °C, 59 °C, 60 °C, 61 °C, 62 °C, 63 °C, the expected PCR product size is 105 bp. Amplification results see Figure 7 , it was found that no target band appeared in the swimming lane, which proved that the L4 primer cannot be used for the detection of PCV1 virus with the L1 primer.

[0071] Taking PCV3 as a sample, the primer L5 primer 5'-ACCCAATAAATACCCCCACC-3'(SEQ ID NO.5) was designed by using primer design software according to the sequence, and the above-mentioned L1 primer was detected by the method in Example 1, and the annealing temperature was 53 °C, 54 °C, 55 °C, 56 °C, 57 °C, 58 °C, 59 °C, 60 °C, 61 °C, 62 °C, 63 °C,...

Embodiment 3

[0072] The detection method of embodiment 3, PCV1 type and PCV3 type

[0073] With PCV1 type and PCV3 type respectively as sample, detect according to the method in embodiment 1, electrophoresis result shows, PCV1 type amplification fragment size is about 901bp, and PCV3 type expected fragment size is about 152bp ( Figure 9 ), as expected.

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Abstract

The invention discloses a multiple PCR detection primer group and kit for rapidly differentiating the PCV1 type from the PCV3 type. The primer group includes primers L1, L2 and L3, wherein L1 represents 5'-ACCAGCGCACTTCGGCAGCGGCAGCA-3', L2 represents 5'-GCATTGTTCACCAGTACCCA-3', and L3 represents 5'-AAGTCCCCTTCCTGCGTCCGCTAATCT-3'. The special kit has very high sensitivity, and minimum detection quantities for the PCV1 type and the PCV3 type are 1*10<-7> ng / mL and 2*10<-7> ng / mL respectively; the specificity is high, and the amplification results of porcine reproductive and respiratory syndromevirus, classical swine fever virus, porcine pseudorabies virus, porcine parvovirus, porcine epidemic diarrhea virus, transmissible gastroenteritis virus and porcine rotavirus are negative; the repeatability is good, the same sample is detected for 3 times at an interval of one month, and the same result is obtained; moreover, the coincidence rate can reach 95% or above compared with methods of virus isolation, IFA and the like, and the primer group and the kit can be widely used in clinical typing detection of the PCV1 type and the PCV3 type.

Description

technical field [0001] The invention relates to a diagnostic technology in the field of veterinary biotechnology, in particular to a multiplex PCR detection primer set and kit for rapidly distinguishing PCV1 and PCV3 types. Background technique [0002] Porcine circovirus (porcine circovirus, PCV) belongs to the genus Circovirus in the family Circoviridae in taxonomy, and is one of the smallest known animal viruses. Virus particles have a diameter of 14-17nm, a 20-hedron symmetrical structure, no envelope, and contain a covalently closed single-strand circular negative-strand DNA. The genome size is about 1.76kb. PCV is quite resistant to external physical and chemical factors. Even in an acidic environment of pH 3 and a high temperature environment of 72°C, it can survive for a period of time. It is not inactivated by the action of chloroform and has no hemagglutination activity. Common PCV has two serotypes, PCV1 and PCV2. Therefore, the existing PCV detection mainly fo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/701C12Q2537/143
Inventor 焦文强徐引弟王治方王克领张青娴朱文豪李海利张彬许峰
Owner INST OF ANIMAL HUSBANDRY & VETERINARY MEDICINE HENAN ACAD OF AGRI SCI
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