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Fluorescence PCR (polymerase chain reaction) kit for detecting yersinia enterocolitica

A technology for enterocolitis and Yersinia, which is applied in the field of real-time fluorescent PCR reagents for detecting Yersinia enterocolitica, can solve problems such as no specific research content, and achieves a simple and fast detection process, high sensitivity, Avoid tedious effects

Inactive Publication Date: 2012-01-25
南宁海关技术中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Researchers use real-time quantitative PCR (polymerase chain reaction) method with TaqMan probes to detect Yersinia enterocolitica in food, but no specific study content

Method used

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  • Fluorescence PCR (polymerase chain reaction) kit for detecting yersinia enterocolitica
  • Fluorescence PCR (polymerase chain reaction) kit for detecting yersinia enterocolitica

Examples

Experimental program
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Effect test

Embodiment 1

[0041] In the Yersinia enterocolitica fluorescent PCR kit of the present invention, the reagents in the kit include a front primer with a nucleic acid sequence of 5'-AATGCTGTCTTCATTTGGAGC-3', a back primer with a nucleic acid sequence of 5'-ATCCCAATCACTACTGACTTC-3', TaqMan probe whose nucleic acid sequence is 5'-CAAGCAAGCTTGTGATCCTCCG-3', PCR amplification buffer, mixed liquid containing DNA polymerase, magnesium ions and four kinds of deoxynucleosides dATP, dCTP, dGTP, dTTP, deionized water and bacteria body concentration of 10 7 Positive standard solution of cfu / ml. The 5'-end label of the TaqMan probe is 6-carboxysuccinimide ester, and the 3'-end label is 6-carboxytetramethylrhodamine; wherein the molar ratio of the front primer, the back primer, and the TaqMan probe 1:1:1; the molar ratio of the four dNTPs is dATP:dCTP:dGTP:dTTP=l:l:l:l. This is the fluorescent PCR kit for detecting Yersinia enterocolitica of the present invention.

Embodiment 2

[0042] Example 2: Quantitative detection of Yersinia enterocolitica in stool samples

[0043] 1. Bacterial DNA Extraction

[0044] (1) Add phosphate buffered saline (PBS) 4 times the amount of the sample and mix well with the stool sample, bathe in 100°C for 15 minutes, centrifuge at 8000 rpm (revolutions per minute) for 5 minutes, take 500 μL of supernatant, add 50 μL of 10% ten Sodium dialkyl sulfate (SDS), 20 μL proteinase K, 50 ° C water bath for 1 h, shake once every 15 min.

[0045] (2) Add an equal amount of phenol, shake well, centrifuge at 10000rpm for 5min.

[0046] (3) Take the supernatant, add 300 μL each of phenol and chloroform, shake well, and centrifuge at 10,000 rpm for 5 minutes.

[0047] (4) Take the supernatant (do not inhale sundries), add an equal amount of chloroform to mix, centrifuge at 10000rpm for 5min.

[0048] (5) Take the supernatant (do not inhale sundries), add 1000 μL of frozen absolute ethanol, add 1 / 10 volume, 3 mol / L NaAc, and set it at -...

Embodiment 3

[0061] Example 3: Quantitative detection of Yersinia enterocolitica in food samples

[0062] 1. Bacterial DNA Extraction

[0063] (1) Add phosphate buffer saline (PBS) 4 times the amount of the sample and grind and homogenize the food sample, bathe at 100°C for 15 minutes, centrifuge at 8000 rpm (revolutions per minute) for 5 minutes, take 500 μL of supernatant, add 50 μL of 10% ten Sodium dialkyl sulfate (SDS), 20 μL proteinase K, 50 ° C water bath for 1 h, shake once every 15 min.

[0064] (2) Add an equal amount of phenol, shake well, centrifuge at 10000rpm for 5min.

[0065] (3) Take the supernatant, add 300 μL each of phenol and chloroform, shake well, and centrifuge at 10,000 rpm for 5 minutes.

[0066] (4) Take the supernatant (do not inhale sundries), add an equal amount of chloroform to mix, centrifuge at 10000rpm for 5min.

[0067] (5) Take the supernatant (do not inhale sundries), add 1000 μL of frozen absolute ethanol, add 1 / 10 volume, 3 mol / L NaAc, and set it a...

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Abstract

The invention relates to a fluorescence PCR (polymerase chain reaction) kit for detecting yersinia enterocolitica, reagents in the kit comprise a front primer, a rear primer, a TaqMan probe, a fluorescence PCR premixed solution, an ROX (roxithromycin) solution, pure water and a positive standard solution, wherein the front primer, the rear primer and the TaqMan probe are DNA (deoxyribonucleic acid) fragments of 20-21 bases, which are synthesized according to a specific and conserved gene nucleotide sequence of the yersinia enterocolitica and can be specifically combined with the yersinia enterocolitica. The invention further provides a method for detecting the yersinia enterocolitica, and the method comprises the following steps: performing pretreatment on samples; using the reagents of the kit for detection; and analyzing the detection result. The kit is simple, fast, sensitive, accurate and suitable for detecting a large number of samples.

Description

technical field [0001] The invention relates to the technical field of real-time fluorescent PCR detection and analysis, in particular to a real-time fluorescent PCR reagent and method for detecting Yersinia enterocolitica. Background technique [0002] Yersinia enterocolitica can cause gastroenteritis, arthritis, sepsis and erythema nodosum in humans and other animals, and is a very important pathogen of zoonotic diseases. The bacteria are widely distributed in our country, and pathogenic bacteria can be isolated from people, animals, external environment and food. The disease is closely related to people's daily life. Epidemiological studies have shown that Yersinia enterocolitica can grow and reproduce at refrigerated temperatures, so refrigerators are an important source of infection for Yersinia enterocolitica, commonly known as "refrigerators". bacteria". [0003] The traditional isolation and identification method for the detection of Yersinia enterocolitica takes a...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/06C12Q1/04G01N21/64
Inventor 盘宝进温和心谢永平
Owner 南宁海关技术中心
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