PCR detection method and kit for francisella tularensis and subspecies of francisella tularensis

A technology of Tularensis and detection reagents, which is applied in the field of PCR detection methods and kits for Tularensis and its subspecies, and can solve the problem of molecular biology detection technology, etc. problem, to achieve the effect of reducing bad data, good specificity, and easy promotion

Active Publication Date: 2019-02-15
SHANDONG BINZHOU ANIMAL SCI & VETERINARY MEDICINE ACADEMY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the methods reported at home and abroad are all the general antigen and antibody detection methods of T. tularensis, and there are no relevant reports on molecular biology detection techniques for the specific detection methods of T. tularensis subspecies.

Method used

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  • PCR detection method and kit for francisella tularensis and subspecies of francisella tularensis
  • PCR detection method and kit for francisella tularensis and subspecies of francisella tularensis
  • PCR detection method and kit for francisella tularensis and subspecies of francisella tularensis

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] The design of embodiment 1 tularemia PCR kit primer

[0052] 1. Materials

[0053] The typical sequences of four T. tularensis subspecies in the NCBI database were selected as follows: AJ1749949, AM233362, AM286280, CP000437, CP000439, CP000915, CP002558, CP003862, CP010115, CP010446, CP025778.

[0054] 2. Method

[0055] Use the database of the American Library of Medicine to compare the above representative sequences (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) to find the specific sequences of each subspecies.

[0056] 3. Results

[0057] Homology comparison results of various subspecies of T. tularensis see Figure 16 ; Specific fragment analysis results are shown in Table 1:

[0058] Table 1

[0059]

[0060] By further analyzing the fragments of unique genes, one unique gene was screened for each subspecies, and the designed primers are shown in Table 2:

[0061] Table 2

[0062]

Embodiment 2

[0063] The construction of embodiment 2 tularensis plasmid standard items

[0064] 1. Materials

[0065] EZ-T Fast Ligation Kit Genstar, 2×Taq PCR StarMix with Loading Dye were purchased from Beijing Kangrun Chengye Biotechnology Co., Ltd.;

[0066] Biospin gel recovery kit: purchased from Hangzhou Bioer Biotechnology Co., Ltd.;

[0067] Templates for 4 subspecies of T. tularensis: provided by the Key Laboratory of Binzhou Animal Husbandry and Veterinary Research Institute, Shandong Province.

[0068] 2. Method

[0069] According to the instructions of 2×Taq PCR StarMix with Loading Dye, use the primers in Table 1 to carry out the target fragments of the 4 subspecies of T. tularensis and the general detection segment of T. tularensis (ie, the target segment amplified by the universal primers, SEQ ID NO: 15) After amplification, 5 fragments were recovered using the Biospin Gel Recovery Kit; the recovered target fragments were ligated according to the instructions of the EZ-T...

Embodiment 3

[0074] Example 3 Tularemia PCR sample detection and method accuracy evaluation

[0075] 1. Materials

[0076] 30 unknown clinical samples from wild rabbits.

[0077] Fluorescent RT-PCR Detection Kit for Tularemia: TaqMan Francisetularensis Detection Kit was purchased from ThermoFisher.

[0078] 2. Method

[0079] (1) Genome extraction

[0080] For the preparation of clinical samples, take suspected disease materials or swabs of sick animals, add an appropriate amount of PBS, and follow-up extraction steps follow the method of bacterial commercial DNA extraction kits to obtain high-purity bacterial genomic DNA, which is stored in - Standby at 20°C.

[0081] Preparation and quantification of positive standard plasmid: Use the primers designed above to amplify the target fragment, connect to EZ-T vector, transform DH5α competent cells, extract and purify the plasmid vector, sequence to confirm the correctness of the inserted fragment, and use OD260 / 280 to quantify , stored...

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Abstract

The invention provides a PCR detection method and kit for francisella tularensis and subspecies of francisella tularensis. The kit contains at least a universal primer (SEQ ID NO:1-2) for detecting francisella tularensis and a specific PCR primer (SEQ ID NO:3-10) for detecting the subspecies of francisella tularensis, namely F.m, F.n, F.Tu and F.h. By adopting the PCR detection method provided bythe invention, francisella tularensis can be quickly determined, the subspecies of francisella tularensis can be determined, an etiology can be directly detected, complicated processes of conventionalbacterial isolation, culture and biochemical identification can be avoided, the detection time is shortened, the operation is convenient, the detection method can be skillfully used after simple training, at the same time, an operator can be avoided from contacting with pathogens directly, thereby improving the safety of an inspection process. The method is high in sensitivity, strong in specificity, and capable of distinguishing the subspecies of francisella tularensis from other subspecies without any cross reaction. The method provides a powerful technical support for the detection of a base layer analysis unit.

Description

technical field [0001] The invention belongs to the technical field of bacteria detection, and in particular relates to a PCR detection method and a kit for tularensis and subspecies thereof. Background technique [0002] At present, domestic and foreign detection methods for tularemia mainly include biochemical detection methods, PCR methods, and fluorescent RT-PCR methods. Among them, biochemical detection methods require the isolation of pathogens. At present, most domestic laboratories do not have the protection conditions for isolation and cultivation; Utilizing the molecular biology method, the existing PCR method and the fluorescent RT-PCR method can quickly carry out the identification of the tularensis. However, there are four subspecies of Tularensis, and their virulence and geographical distribution are different. The distribution and virulence of the four subspecies of Tularensis (Francisella genus) are as follows: Francisellatularensis subsp.Tularensis, F.tu, th...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11C12R1/01
CPCC12Q1/686C12Q1/689C12Q2600/16
Inventor 李书光何诚沈志强曲光刚杨丽芳张娜赵家磊
Owner SHANDONG BINZHOU ANIMAL SCI & VETERINARY MEDICINE ACADEMY
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