Primer, probe and kit for detecting canine parvovirus and detection method

A technique for detection of canine parvovirus and primers, applied in biochemical equipment and methods, measurement/testing of microbes, DNA/RNA fragments, etc., can solve unfavorable large sample size detection and epidemiological investigations, complex extraction steps And strict, poor accuracy of test strips and other problems, to achieve the effect of small operation error, meet clinical testing and epidemic monitoring, and simple steps

Inactive Publication Date: 2016-07-27
中国人民解放军成都军区疾病预防控制中心
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AI Technical Summary

Problems solved by technology

[0004] At present, the method of detecting canine parvovirus is mainly to observe clinical symptoms. There are test strips for virus detection methods, and there are also reports that traditional PCR and fluorescent quantitative PCR are used, but the accuracy of test strips is generally poor, and the traditional PCR method takes a long time. and low sensitivity
[0005] However, although the current fluorescent quantitative PCR has improved sensitivity and accuracy compared with the traditional PCR method and test strip detection method, it still has defects such as high detection cost, long time-consuming, and complicated operation.
For example, in fluorescent quantitative PCR, it is necessary to use nucleic acid extraction sol

Method used

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  • Primer, probe and kit for detecting canine parvovirus and detection method
  • Primer, probe and kit for detecting canine parvovirus and detection method
  • Primer, probe and kit for detecting canine parvovirus and detection method

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Experimental program
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Effect test

Embodiment 1

[0058] 1. Design of specific primers and probes

[0059] According to the nucleic acid sequence of canine parvovirus retrieved from Genbank, primers and probes for detecting canine parvovirus were designed, and its sequence is as follows:

[0060] Upstream primer (CPV-F): 5'-GAAGGTATAAATTCACCAGGTTGC-3', as shown in SEQ ID NO.1;

[0061] Downstream primer (CPV-R): 5'-GTGCAAGGTCCACTACGTCC-3', as shown in SEQ ID NO.2;

[0062] Fluorescent probe (CPV-probe): 5'FAM-AGACACAAGCGGCAAGCAATCCTC-3'BHQ-1.

[0063] The above primers and fluorescent probes were synthesized by Shanghai Gemma Pharmaceutical Technology Co., Ltd.

[0064] The concentrations of upstream primers, downstream primers and fluorescent probes were 0.2 μM, 0.2 μM and 0.2 μM, respectively.

[0065] The length of the target fragment amplified by the above-mentioned primers and probes is 112 bp.

[0066] 2. Components of the kit

[0067] The kit of the present invention includes four components: a reaction solution A...

Embodiment 2

[0098] Embodiment 2 Sensitivity experiment

[0099] Take the isolate of canine parvovirus preserved by the Center for Disease Control and Prevention of Chengdu Military Region (the isolate can be obtained by the public). The reference information of the isolate of canine parvovirus refers to the literature: Isolation and identification of canine parvovirus, Du Qiang, Qiu Wei, Fan Quanshui, Liu Hua Li Zuosheng, Zheng Ying, and Zhang Fuqiang, Vol. 30, No. 3, 2009, pages 55-58. Virus titer titrated TCID50 to 10 -4 , followed by 10 times of dilution, the dilution times are: 1 times, 10 times, 100 times, 1000 times, 10000 times, 100000 times, 1000000 times, the diluted solution is recorded as 10 times 0 、10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 , carry out sensitivity experiment, specific experiment procedure with reference to above-mentioned embodiment 1.

[0100] The results of one-step CPV fluorescence detection sensitivity experiment are as follows: figure 1 , the ampl...

Embodiment 3

[0107] Embodiment 3 specificity experiment

[0108] According to the detection method of the above-mentioned embodiment 1, the canine parvovirus, canine distemper virus, and canine adenovirus-II strains isolated from animal specimens are the samples to be tested, and the above-mentioned samples to be tested are all stored in the Center for Disease Control and Prevention of the Chengdu Military Region The laboratory can be obtained by the public. For specific detailed information, please refer to the literature: Cloning and Expression of H Gene of Canine Distemper Virus Yunnan Strain, Nie Fuping, Li Zuosheng, Qiu Wei, Zhang Fuqiang, Zhang Lianjiang, Song Guiqiang, Long Guiwei, Liao Jin, Ren Yuying, Yu Fang, Zhang Quanpeng, Wang Lingqiang, Fan Quanshui, Advances in Veterinary Medicine, Vol. 29, No. 1, 2008: pp. 2-12. Experimental Immunization Research of CDV, CPV, CAV Triple DNA Vaccine, Zheng Ying, Qiu Wei, Li Lihong, Nie Fuping, Long Guiwei, Li Zuosheng, Zhang Fuqiang, Li Jian...

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Abstract

The invention relates to a primer, probe and kit for detecting canine parvovirus and a detection method.According to the fluorogenic quantitative PCR primer for detecting canine parvovirus, an upstream detection primer CPV-F has the nucleotide sequence as shown in the SEQ ID NO.1, and a downstream detection primer CPV-R has the nucleotide sequence as shown in the SEQ ID NO.2.The probe has the nucleotide sequence as shown in the SEQ ID NO.3.The kit comprises the primer and the probe.The fluorogenic quantitative PCR primer, probe and kit for detecting canine parvovirus have the advantages that operation is easy, cost is low, sensitivity is high and specificity is high, and can guarantee clinical detection.

Description

technical field [0001] The invention belongs to the field of virus detection, in particular to a primer, a probe, a kit and a detection method for fluorescent quantitative PCR for directly detecting canine parvovirus in one step. Background technique [0002] Canine parvovirus (Canine Parvovirus, CPV) is a severe infectious virus that causes canine animals with severe vomiting, hemorrhagic enteritis, non-suppurative myocarditis and significant reduction of white blood cells. Canine parvovirus is a highly contagious, highly contagious disease characterized by vomiting, diarrhea, and leukopenia. Cases of natural infection with canine parvovirus occur frequently, even after CPV vaccine immunization, and the mortality rate of puppies is very high (20%-30%). With the rapid development of the dog breeding industry, especially the substantial increase in the number of experimental dogs and pet dogs, the parvovirus infection of dogs is becoming more and more serious, which has caus...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/701C12Q1/686C12Q2563/107C12Q2545/114C12Q2531/113C12Q2545/113
Inventor 王文博邱薇曹雪峰弓超陈锚锚郭平杨显君范泉水
Owner 中国人民解放军成都军区疾病预防控制中心
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