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Vibrio cholerae multiplex fluorescence PCR detection kit as well as preparation and application thereof

A detection kit, Vibrio cholerae technology, applied in the field of biotechnology detection, can solve the problems of poor accuracy, cross-contamination, low efficiency, etc., and achieve the effect of simple operation, high sensitivity, and improved detection efficiency

Active Publication Date: 2016-05-25
广东省疾病预防控制中心 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In molecular biological detection methods, although common PCR method detects Vibrio cholerae with high sensitivity and specificity, it can only detect one gene at a time, and the efficiency is low; when the amplified product is analyzed by gel electrophoresis, It is easy to cause cross-contamination; electrophoresis results often need to be judged subjectively by experimenters, and the accuracy is not good

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  • Vibrio cholerae multiplex fluorescence PCR detection kit as well as preparation and application thereof
  • Vibrio cholerae multiplex fluorescence PCR detection kit as well as preparation and application thereof
  • Vibrio cholerae multiplex fluorescence PCR detection kit as well as preparation and application thereof

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Embodiment 1

[0043] The preparation of embodiment 1 kit

[0044] The primers and probes for Vibrio cholerae hemolysin gene detection, the primers and probes for Vibrio cholerae O1 antigen gene detection, the primers and probes for Vibrio cholerae O139 group antigen gene detection, the primers and probes for Vibrio cholerae enterotoxin gene detection were synthesized respectively. needle, its nucleotide sequence is shown in Table 1 below:

[0045] Table 1

[0046]

[0047] The above-mentioned sets of primer pairs and probes can be packaged individually, or can be combined to make a multiple fluorescent PCR detection mixture. In the multiplex fluorescent PCR detection mixture, the amounts of the above-mentioned primers and probes can be conventional amounts known to those skilled in the art.

[0048] That is to say, the kit of the present invention may contain the aforementioned independently packaged sets of primer pairs and probes, or may contain a configured multiplex fluorescent PCR...

Embodiment 2

[0050] Sensitivity evaluation of embodiment 2 kit

[0051] Experiment purpose: determine the detection limit (minimum detection concentration) of the present invention (embodiment 1) multiple fluorescent PCR kit

[0052] experimental method:

[0053] 1. Preparation of positive control substance:

[0054] The pMD8 plasmid containing Vibrio cholerae hemolysin, Vibrio cholerae O1, O139 group-specific antigens and specific amplified fragments of enterotoxin gene was constructed as a positive control. The pMD8 plasmid is connected with Vibrio cholerae hemolysin gene detection primers, Vibrio cholerae O1 group antigen gene detection primers, Vibrio cholerae O139 group antigen gene detection primers, and Vibrio cholerae enterotoxin gene detection primers. The augmenter fragment can be jointly recognized by the above-mentioned 4 kinds of primers and probes. It was verified by sequencing that the positive plasmid was successfully constructed.

[0055] Wherein, the nucleotide sequen...

Embodiment 3

[0081] Example 3 Kit Test Result Specificity Evaluation

[0082] Experimental purpose: Select the nucleic acid extract of Vibrio cholerae sample that has been verified by Sanger sequencing to test the specificity and reliability of multiple fluorescent PCR detection results.

[0083] experimental method:

[0084] 1. Sample processing: select nucleic acid extraction solutions of different types of samples from the specimen bank, thaw at room temperature and take 4ul for subsequent experiments.

[0085] Feces sample: Pick the feces the size of a grain of rice, put them into a centrifuge tube that has been pre-added with 0.5ml of normal saline, shake and mix, centrifuge at 13000rpm for 2 minutes, and remove the supernatant; add 100ul of the nucleic acid extractor provided in the kit to the precipitate extract, mix well. Boiling water bath for 10 minutes, centrifugation at 13000rpm for 5 minutes, the supernatant is the sample nucleic acid extraction solution, which can be direct...

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Abstract

The invention belongs to the field of biotechnological detection and particularly relates to a vibrio cholerae multiplex fluorescence PCR detection kit as well as preparation and application thereof. The kit provided by the invention comprises a vibrio cholerae hemolysin gene detection primer and a probe, a vibrio cholerae O1 antigen gene detection primer and a probe, vibrio cholerae O139 antigen gene detection primer and a probe and a vibrio cholerae enterotoxin gene detection primer and a probe. The kit has high detection sensitivity, the lowest detection limit is 1*10<3>copy / ml, and the accuracy and positive rate both reach 100%.

Description

technical field [0001] The invention belongs to the field of biotechnology detection, and in particular relates to a vibrio cholerae multiple fluorescent PCR detection kit and its preparation and application. Background technique [0002] Cholera is a severe intestinal infectious disease and one of the legally notifiable infectious diseases in my country. The pathogen is Vibrio cholerae (Vibriocholerae). Cholera has a sudden onset and rapid spread. Patients have severe diarrhea and vomiting symptoms, which can lead to a series of severe syndromes such as dehydration and shock. Those who are not diagnosed and treated in time have a high mortality rate. [0003] At present, Vibrio cholerae can be divided into more than 210 serogroups according to different bacterial antigens, of which only O1 and O139 groups can cause cholera epidemics. O1 group is divided into classical biotype (Classicalbiotype) and El Tor biotype (ElTorbiotype). There have been 7 worldwide pandemics of ch...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/686C12Q1/689C12Q2600/16C12Q2537/143C12Q2563/107C12Q2545/113Y02A50/30
Inventor 柯昌文李柏生柯碧霞肖红邵俊斌朱勤玮王凯熊磊李鸿雁
Owner 广东省疾病预防控制中心
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