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Specific primer of cattle mini cryptosporidium and PCR (polymerase chain reaction) detection method and application of specific primer

A technology of Cryptosporidium parvum and a detection method, applied in the field of parasite detection, can solve the problems of time-consuming, labor-intensive, complicated operation, etc., and achieve the effects of low price, accurate results and good primer specificity

Inactive Publication Date: 2020-05-08
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method is cumbersome to operate and requires two rounds of PCR amplification, which is time-consuming and labor-intensive, and requires the use of sequencing technology for species identification

Method used

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  • Specific primer of cattle mini cryptosporidium and PCR (polymerase chain reaction) detection method and application of specific primer
  • Specific primer of cattle mini cryptosporidium and PCR (polymerase chain reaction) detection method and application of specific primer
  • Specific primer of cattle mini cryptosporidium and PCR (polymerase chain reaction) detection method and application of specific primer

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Experimental program
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Embodiment 1

[0052] Establishment of conventional PCR and nano-PCR detection methods for Cryptosporidium parvum in cattle

[0053] 1. Primer design and synthesis:

[0054] Primers were designed with reference to the Cryptosporidium parvum Iowa II scully gene locus (Gene ID: 3373948) published in GenBank. The reference sequence is as follows:

[0055]

[0056]

[0057] Using the software DNAMAN 7, according to the sequence of the Cryptosporidium parvum Iowa II scully gene (Gene ID: 3373948), the specific primers for the Cryptosporidium parvum Iowa II scully gene were screened and synthesized. Synthesis, primer sequences are listed in Table 1.

[0058] Table 1 Specific primers for PCR amplification of Cryptosporidium parvum

[0059]

[0060] Second, the establishment of common PCR method

[0061] (1) The general PCR reaction system is preset to 10×ex Taq buffer (Mg 2+ free) 1.25 μL, MgCl 2 (25mM) 1μL, dNTP (2.5mM) 1μL, upstream and downstream primers (10mM) 0.5μL each, ex Taq e...

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Abstract

The invention relates to the technical field of parasite detection and in particular relates to a specific primer of cattle mini cryptosporidium and a PCR (polymerase chain reaction) detection methodand application of the specific primer. By adopting the method, mini cryptosporidium in a sample can be rapidly, sensitively and specifically detected. Sequences of the specific primer of the cattle mini cryptosporidium are: an upstream primer CpF: 5'-AGTGGTTACAGGTGGGATGAGT-3', and a downstream primer CpR: 5'-GCGAGTTTCCTTGATTCATAGC-3'. The common PCR and nano PCR detection method of the primer comprise the following steps: firstly, extracting DNA (deoxyribonucleic acid) of the mini cryptosporidium from a sample to be detected; by taking the DNA as a template, performing PCR amplification by using the specific primer, and performing electrophoresis detection on the amplification product; and taking the conclusion that if a specific band of about 410bp shows in electrophoresis results, it means that the mini cryptosporidium exists in the sample.

Description

[0001] 1. Technical field: [0002] The invention relates to the technical field of parasite detection, in particular to a specific primer for cryptosporidium parvum and its PCR detection method and application. [0003] 2. Background technology: [0004] Cryptosporidiosis in cattle is a serious parasitic disease caused by Cryptosporidium parasitizing on the epithelial cells of the gastrointestinal tract of cattle. The study found that there are four common Cryptosporidium species in cattle, namely Cryptosporidium parvum (C. Insects (C. ryanae). Among them, Cryptosporidium parvum is an important zoonotic species, which seriously threatens the health of humans and domestic animals. In my country, the infection rate of C.parvum in pre-weaning dairy cows can be as high as 31.4% in some areas, and its infection can cause severe watery diarrhea in calves, leading to severe dehydration and even death. Infected adult cattle will also have short-term weight loss and impaired product...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6893C12Q1/686C12Q1/04C12N15/11C12R1/90
CPCC12Q1/6893C12Q1/686C12Q2563/155C12Q2563/137
Inventor 王钰鑫王一许丹娜宋军科赵光辉李媛董和平
Owner NORTHWEST A & F UNIV