Universal real time fluorescent PCR detection method of trichinella

A real-time fluorescence and detection method technology, applied in the field of PCR amplification primers and probes, can solve the problems of high missed detection rate of inspection and quarantine methods, high subjective dependence of testers, etc., and achieve the effect of wide linear range and good stability

Inactive Publication Date: 2009-03-11
NORTHEAST AGRICULTURAL UNIVERSITY +1
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Problems solved by technology

[0009] Since there are multiple isolated species of Trichinella spiralis pathogens, conventional inspection and quarantine methods have a high rate of missed detection, and the subjective dependence on the tester is high. Therefore, it is urgent to establish a specific, sensiti

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  • Universal real time fluorescent PCR detection method of trichinella
  • Universal real time fluorescent PCR detection method of trichinella
  • Universal real time fluorescent PCR detection method of trichinella

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[0041] The present invention will be further described below in conjunction with specific embodiments:

[0042] 1. Preparation of PCR template: Refer to the instructions of TIANamp Genomic DNAKit for DNA extraction and purification. Add various reagents in proportion.

[0043] 1.1 Take an appropriate amount of the sample to be tested, add 200μl of buffer GA, and add 20μl of proteinase K (20mg / ml), in a water bath at 56°C for 2h;

[0044] 1.2 Add 200μl of buffer GB and place it in a 70℃ water bath for 10 minutes, then centrifuge briefly;

[0045] 1.3 Add 200μl of absolute ethanol, after a brief centrifugation, transfer the supernatant to the spin column CB3, centrifuge at 12000r / min for 1min, discard the liquid in the collection tube;

[0046] 1.4 Add 500μl of protein-removing solution to the spin column CB3, centrifuge at 12000r / min for 1min, and discard the liquid in the collection tube;

[0047] 1.5 Add 700μl of rinsing solution PW to the spin column CB3, centrifuge at 12000r / mi...

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Abstract

The invention provides a trichina general real-time fluorescent PCR detection method. Conserved region sequences of LS-rRNA genes of mitochondrion of various trichina isolated species are determined to be amplified target sequences; and a pair of special primers and a TaqMAN MGB probe are designed, synthesized and applied to general detection of trichina. As shown by a quantitative standard curve manufactured by positive quality-control standard products with different concentration gradients, the logarithm value and the Ct value of the quantitative die plate number with the different gradients have good relativity, and the correlation coefficient is 0.9984; simultaneously, the detection method has high sensitivity, and 132 copy numbers and 10<-5> trichinas can be detected at the lowest; as shown by detection of other parasites, health hosts and water contrast, the method has good specificity; and as shown by the repeated tests, the detection system provided by the invention has good stability, and the detection result has small variation and is controlled within the statistic range.

Description

(1) Technical field [0001] The invention relates to a primer and a probe for PCR amplification of mitochondrial Ls-rRNA gene fragments of each isolated species of Trichinella spiralis, and a general detection method for simultaneously performing isolated species of Trichinella spiralis. (2) Background technology [0002] Trichinosis (Trichinosis) is an important zoonotic parasitic disease, which is mainly infected by eating raw or half-cooked pork or other animal meat containing Trichinella larva cysts. Due to its extremely poor host specificity and the diversity of pathogen transmission routes, almost all mammals can be infected. Both carnivores, omnivores, insectivores, and herbivores can be infected, and even some birds can be infected. Humans are also one of the susceptible hosts of Trichinella spiralis. So far, at least more than 150 kinds of mammals have been reported to be infected with Trichinella spiralis, common ones are humans, pigs, rats, dogs, bears, foxes, wol...

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64
Inventor 宋铭忻张子群袁金钱谢晓峰路义鑫李维刚韩彩霞由轩罗公平
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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