Enterotoxigenic escherichia coli heat stable enterotoxin gene SYBR Green I fluorescent quantitative PCR diagnostic kit

A technology of heat-resistant enterotoxin and diagnostic kit, which is applied in the directions of microorganism-based methods, microbial determination/examination, DNA/RNA fragments, etc.

Inactive Publication Date: 2018-06-19
GUIZHOU INST OF ANIMAL HUSBANDRY & VETERINARY
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  • Application Information

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Problems solved by technology

However, there is no PCR diagnostic kit for rapid and accurate diagnosis of the heat-resistant enterotoxin gene of pig-derived enterotoxigenic E. coli in the prior art, so that the diagnosis of the pig-derived enterotoxigenic E. And detection still has defects such as cumbersome operation, low sensitivity, and can only be qualitative but not quantitative

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  • Enterotoxigenic escherichia coli heat stable enterotoxin gene SYBR Green I fluorescent quantitative PCR diagnostic kit
  • Enterotoxigenic escherichia coli heat stable enterotoxin gene SYBR Green I fluorescent quantitative PCR diagnostic kit
  • Enterotoxigenic escherichia coli heat stable enterotoxin gene SYBR Green I fluorescent quantitative PCR diagnostic kit

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Embodiment Construction

[0069] Embodiment of the present invention: an enterotoxigenic Escherichia coli heat-resistant enterotoxin gene SYBR Green I fluorescent quantitative PCR diagnostic kit includes 40 μL of specific primers, 250 μL of fluorescent quantitative PCR reaction mixture (Premix Ex Taq), 20 μL of ROX Reference Dye, 20 μL negative control, 20 μL positive control, 500 μL ultrapure water; wherein, the specific primers include upstream primers and downstream primers, and the upstream primers are

[0070] 5',-TATCAATAGCATTCAGCACCAT-3', the downstream primer is

[0071] 5', -AATGTCCGTCTTGCGTTAGGA-3'.

[0072] In some embodiments, the negative control is ultrapure water; the positive control is pMD-18T-ST plasmid.

[0073] In some embodiments, the specific primer concentration is selected as 10 μmol / L. For the specific primers, the amount of upstream primers and downstream primers is equal.

[0074] In certain embodiments, the specific primer has an annealing temperature of 55°C.

[0075] I...

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Abstract

The invention an enterotoxigenic escherichia coli heat stable enterotoxin gene SYBR Green I fluorescent quantitative PCR diagnostic kit, and relates to the technical field of kits. The enterotoxigenicescherichia coli heat stable enterotoxin gene SYBR Green I fluorescent quantitative PCR diagnostic kit comprises 40 mu L of specific primers, 250 mu L of fluorescent quantitative PCR reaction mixed liquor (Premix EX Taq), 20 mu L of ROX Reference Dye, 20 mu L of negative control, 20 mu L of positive control and 500 mu L of ultrapure water. A pair of special primers is designed according to pig source enterotoxigenic escherichia coli (ETEC) heat stable enterotoxin gene sequence (STII) in GenBank to perform a test, and the test method has sensitivity of 1.0*10<1> copies/mu L, is good in repeatability; and PCR product sequencing results are compared with the ST gene sequence in GenBank, and similarity is 98.2%-100%. The kit has the characteristics of being high in detecting speed and high indetecting rate.

Description

technical field [0001] The invention relates to the technical field of kits, in particular to a diagnostic kit for enterotoxigenic Escherichia coli heat-resistant enterotoxin gene SYBRGreen I fluorescent quantitative PCR. Background technique [0002] Enterotoxigenic Escherichia coli (ETEC) is an important pathogen causing diarrhea in piglets. Newborn piglets often die due to severe watery diarrhea and rapid dehydration after being infected by ETEC. The main pathogens of piglet diarrhea caused by ETEC are host-specific adhesins (F4, F5) and enterotoxins (Yuan Wanzhe, He Kongwang, Lu Chengping, et al. Research progress on major virulence factors of enterotoxigenic Escherichia coli[J], Advances in Veterinary Medicine, 2005, 26(2):6-9). ETEC is adsorbed to small intestinal epithelial cells through the adhesin on the bacterial surface, and then colonizes and proliferates in the small intestine (SommerU, Petersen J, Pfeiffer M, Schrotz-King P, Morsczeck C.2010.Comparison of surf...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/10C12N15/11C12R1/19
CPCC12Q1/686C12Q1/689C12Q2600/158C12Q2563/107C12Q2545/114
Inventor 姜玲玲杨莉杨丽娟唐远江卢昱希徐景娥史开志余波
Owner GUIZHOU INST OF ANIMAL HUSBANDRY & VETERINARY
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