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Kit for detection of Yersinia pestis through three-color fluorescent quantification PCR and detection method

A Yersinia, fluorescence quantitative technology, applied in the biological field, can solve the problems of difficult template preparation, cumbersome operation, difficult to automate, etc.

Inactive Publication Date: 2014-02-19
潘光合 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the conventional PCR method is widely used, mostly targeting the caf1 gene on the pMT1 plasmid and the pla gene on the pPCP1 plasmid encoding the F1 antigen of Yersinia pestis. Missed detection; the template preparation of competitive PCR is difficult, and the point variation of a single positive quality control has a great impact on the results, which is difficult to automate; the real-time fluorescent quantitative PCR method, the whole process of amplification and product analysis is closed in a single tube It is carried out under certain conditions and controlled by a microcomputer to realize real-time dynamic detection of PCR amplification products and automatic analysis of results without post-processing of PCR products. This method is easier and faster to operate, but the current real-time fluorescence quantitative The PCR detection kit for Yersinia pestis can only detect a single target gene. If multiple target genes are detected, multiple amplifications are required, and the operation is cumbersome

Method used

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  • Kit for detection of Yersinia pestis through three-color fluorescent quantification PCR and detection method
  • Kit for detection of Yersinia pestis through three-color fluorescent quantification PCR and detection method
  • Kit for detection of Yersinia pestis through three-color fluorescent quantification PCR and detection method

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Experimental program
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Effect test

Embodiment 1

[0050] 1. Sample Collection and Pretreatment

[0051] Take 2ml of femoral artery blood from the rats to be tested, put it in an anticoagulant tube containing sodium citrate, mix well, add Herbella enrichment solution for culture and enrichment.

[0052] 2. Extraction of DNA

[0053] Take 1ml of the enrichment solution and put it into a clean 1.5ml centrifuge tube, centrifuge at 13,000rpm for 2min, discard the supernatant, add 50μl of nucleic acid extraction solution to the pellet, mix well, then boil at 100°C for 10min, centrifuge at 12,000rpm for 2min, the supernatant is ready for the DNA to be tested.

[0054] 3. Three-color fluorescent quantitative PCR amplification

[0055] Each test reaction system was prepared as follows: 21 μl of three-color PCRMIX, 1 μl of Taq enzyme system, and centrifuged briefly. Then add 3 μl of DNA to be detected; set up positive and negative control substances according to the above system, and add 3 μl of positive quality control substance or...

Embodiment 2

[0059] 1. Sample Collection and Pretreatment

[0060] The liver, spleen, lung and lymph nodes of the rats were collected from diseased tissues with a large amount of bacteria, put into sterile tubes, and sealed for inspection.

[0061] 2. Extraction of DNA

[0062] Take about 100mg of tissue and transfer it to a 1.5ml clean centrifuge tube, crush it, add 50μl nucleic acid extraction solution, mix thoroughly, then boil at 100°C for 10min, centrifuge at 12,000rpm for 2min, the supernatant is the DNA to be tested.

[0063] 3. Three-color fluorescent quantitative PCR amplification

[0064] Each test reaction system was prepared as follows: 21 μl of three-color PCR MIX, 1 μl of Taq enzyme system, and centrifuged briefly. Then add 3 μl of DNA to be detected; set up positive and negative control substances according to the above system, and add 3 μl of positive quality control substance or negative control substance. Put each reaction tube into the reaction tank of the real-time f...

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Abstract

The invention relates to a kit for detection of Yersinia pestis nucleic acids and a detection method, and belongs to the biological technology field. The reagents comprises a nucleic acid extracting solution, a Taq enzyme system, fluorescent quantification PCR MIX, positive quality controls and negative reference substances. The detection method employs the three-color fluorescent quantification PCR technology, and through one amplification test, pla and caf1 genes in the plasmids and 3a gene in the chromosomes of Yersinia pestis in samples can be detected qualitatively and quantitatively at the same time. The method comprises the following steps: pretreatment of samples; extraction of nucleic acids; three-color fluorescent quantification PCR detection; analysis of detection results; determination of existence or not of Yersinia pestis in samples to be detected and precise quantification. The detection kit and method have high sensitivity and good specificity, and the method is rapid and efficient. The purpose of rapid detection of Yersinia pestis can be achieved. (The amplification curves of detection of pla gene through three-color fluorescent PCR are shown in the abstract drawings.

Description

1. Technical field [0001] The present invention relates to a kind of using three-color fluorescent quantitative PCR technology, in one experiment simultaneously detects three kinds of specific genes of Yersinia pestis (hereinafter referred to as Yersinia pestis) kit, and further relates to the detection method using this kit, Belongs to the field of biotechnology. 2. Background technology [0002] Plague is caused by the Yersinia pestis bacillus and is one of the most serious severe infectious diseases harmful to human beings. It has the characteristics of strong infectivity, rapid spread, serious illness, and high fatality rate. It is an international quarantine infectious disease. The "Law on the Prevention and Control of Infectious Diseases" promulgated by my country defines plague as a Class A infectious disease. In history, there have been three pandemics of plague, which have affected almost all countries and caused the death of hundreds of millions of people. Among ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/01
CPCC12Q1/04C12Q1/686C12Q2563/107C12Q2531/113
Inventor 潘光合谭勇吴文旺覃杨光梁跃波蓝翊文梁亮甘洁刘健翊徐贵峰曾爱英方晋张凤芬林洁梁也黄少宁万道正梁中平杨会敏
Owner 潘光合