Double-PCR (polymerase chain reaction) detection kit and method for giardia and cryptosporidia of sheep

A technology for Cryptosporidium parvum and Giardia, which is applied in biochemical equipment and methods, microbial measurement/testing, and resistance to vector-borne diseases, etc. It can solve the problems of unsatisfactory detection, low sensitivity, and poor specificity. Achieve the effect of convenient observation, high sensitivity and easy distinction

Inactive Publication Date: 2018-11-13
JILIN UNIV
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Problems solved by technology

Therefore, sheep lack a diagnostic method with high accuracy, convenient and simple operation, strong specificity, good sensitivity, and short cycle that can be used to detect Giardia and Cryptosporidium in large-scale samples. The detection methods of Giardia intestinalis include microscopy, IFA (immunofluorescence assay), in vitro cell infection, in situ hybridization technology, RT-PCR technology, etc. At present, these detection technologies are time-consuming, laborious, inaccurate, low sensitivity, Poor specificity, unable to meet the requirements for detecting feces of mildly infected hosts

Method used

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  • Double-PCR (polymerase chain reaction) detection kit and method for giardia and cryptosporidia of sheep
  • Double-PCR (polymerase chain reaction) detection kit and method for giardia and cryptosporidia of sheep
  • Double-PCR (polymerase chain reaction) detection kit and method for giardia and cryptosporidia of sheep

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Embodiment 1

[0040] Specific gene selection for Giardia duodenum and Cryptosporidium parvum

[0041] Species-specific diagnostic sequences of Giardia duodenum and Cryptosporidium parvum: select the highly conserved TPI gene and 18S rRNA gene sequence, and compare them in NCBI as unique to Giardia and Cryptosporidium parvum, and match the species Specific detection research standards;

[0042] Primer Design for Dual PCR Detection Kit

[0043] Use biological software to design specific diagnostic primers as follows:

[0044] Giardia duodenum PCR primers:

[0045] F1: 5'-CGACCTGACTATGACCAACTC-3';

[0046] R1: 5'-GTCCTGAAGCATTCTCAACACT-3';

[0047] Cryptosporidium parvum PCR primers:

[0048] F2: 5'-CCTTACTCCTTTCAGCACCTTATG-3';

[0049] R2: 5'-TGTTACGACTTCTCCTTCCTCTAA-3';

[0050] Components of the Duplex PCR Detection Kit

[0051] (1) PCR reaction solution: 2.5mM dNTPs, upstream and downstream primers at a final concentration of 10pmol / μL, 10×PCR buffer, rTaq DNA polymerase, DNA templ...

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Abstract

The invention discloses a double-PCR (polymerase chain reaction) detection kit and method for giardia and cryptosporidia of sheep. According to parts of conserved sequences of giardia duodenalis TPI genes and cryptosporidium parvum 18S rRNA genes, a primer is designed, a double-PCR detection method is built, sensitivity and accuracy of the method are improved by optimizing reaction conditions suchas annealing temperature of PCR reaction, amplification products are implemented by 1.0% of agarose gel electrophoresis, and a result is conveniently and rapidly observed. Distinguishing of fragmentswith different targets is one of keys of building the double-PCR detection method, two target fragments amplified by the method have size differences in agarose gel electrophoresis and are easily distinguished, the sensitivity can reach 1*10<3>/g and is higher than that of a reported microscopy method and a conventional PCR detection method of giardia duodenalis and cryptosporidium parvum, and the method is suitable for detection of samples in clinical practice. The detection method can simultaneously and rapidly detect the giardia duodenalis and the cryptosporidium parvum of the sheep and has the advantages of simplicity in operation, high sensitivity, high specificity and the like, and technical support is provided for quality standards of real experiment sheep.

Description

technical field [0001] The invention provides a method for simultaneously detecting sheep Giardia duodenum ( Giardia duodenalis, G. duodenalis ), Cryptosporidium parvum ( C. parvum) The double PCR detection method, the invention also provides the preparation method and usage of the kit, belonging to the technical field of parasite detection. Background technique [0002] Giardia ( Giardia ) and Cryptosporidium ( Cryptosporidium ) is an important zoonotic protozoan, which infects a wide range of hosts, and the clinical symptoms after infection are mainly diarrhea, and cause huge economic losses to animal husbandry and breeding. Cryptosporidium parvum ( C. parvum ) is a tiny zoonotic parasite that exists widely in a variety of vertebrates and parasitizes humans and most mammals. The disease it causes is cryptosporidiosis, mainly diarrhea, and can be life-threatening in severe cases . Giardia duodenum is an important zoonotic parasite. It parasitizes the gallbladder...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6893C12Q1/686
CPCC12Q1/686C12Q1/6893C12Q2600/16C12Q2537/143C12Q2565/125Y02A50/30
Inventor 宫鹏涛张西臣高宇航李显赫任文陟李建华杨举
Owner JILIN UNIV
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