Method for detecting Francisella tularensis using multiple PCR technology

A kind of technical detection, Tura technology, applied in the direction of biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of inability to meet rapid detection, long detection time, low sensitivity, etc.

Inactive Publication Date: 2011-06-15
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] The commonly used detection methods for Francisella tularensis are mostly traditional physiological and biochemical tests, serum antibody detection, enzyme-linked immunosorbent assay (ELISA) and ordinary PCR, etc., but the detection time is long and the sensitivity is low. Obviously, in the face of sudden biological attack, none of the above methods can meet the needs of rapid detection

Method used

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  • Method for detecting Francisella tularensis using multiple PCR technology
  • Method for detecting Francisella tularensis using multiple PCR technology
  • Method for detecting Francisella tularensis using multiple PCR technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Embodiment 1: PCR detection of Francisella tularensis in soil

[0070] 1. Sample pretreatment

[0071] Two soil samples (1g / part) were collected and added to 30mL TSB medium, one of which was inoculated with a single colony of Francisella tularensis F001, and cultured at 200rpm / min at 37°C for 6 hours for pre-enrichment. After enrichment, let it stand, and after the soil sample was precipitated, take 1 μL of the bacterial suspension as a bacterial template for multiplex PCR detection.

[0072] 2. PCR reaction

[0073] PCR reaction system

[0074] Reaction system (20μL):

[0075] 10×PCR Buffer: 2μL,

[0076] d NTP: 2 μL,

[0077] Template: 1 μL,

[0078] Taq enzyme: 0.1 μL,

[0079] Primers: lpxE(+) 0.5 μL, lpxE(-) 0.5 μL,

[0080] lpxF(+) 0.5 μL, lpxF(+) 0.5 μL,

[0081] flmK(+) 1.5 μL, flmK(-) 1.5 μL,

[0082] wxya 2 O: Make up 20 μL.

[0083] PCR reaction process

[0084] The multiplex PCR amplification program and the melting temperature program for...

Embodiment 2

[0093] Embodiment 2: PCR detection of Francisella tulara in water

[0094] 1. Sample pretreatment

[0095]Two water samples (5 mL / part) were collected and 30 mL of TSB medium was added, one of which was inoculated with a single colony of Francisella tularensis F001, and cultured at 200 rpm / min at 37°C for 6 hours for pre-enrichment. After enrichment, 1 μL of the bacterial suspension was used as a bacterial template for multiplex PCR detection.

[0096] 2. PCR reaction: the reaction conditions and system are the same as in Example 1.

[0097] 3. Test results and description

[0098] Analyzed by 1% agarose gel electrophoresis, the results are shown in the appendix Figure 5 .

[0099] The results showed that the corresponding specific bands were amplified in the water samples added with bacteria (see attached Figure 5 4th track), and no corresponding bands appeared in the samples without artificial bacteria addition (see attached Figure 5 track 5).

Embodiment 3

[0100] Embodiment 3: the PCR detection of Francisella tularensis in the air

[0101] 1. Sample pretreatment

[0102] Two samples of dust in the air (1 g / part) were collected with a vacuum cleaner, and 30 mL of TSB medium was added, one of which was inoculated with a single colony of Francisella tularensis F001, cultured at 37°C, 200 rpm / min for 6 hours for pre-enrichment. After enrichment, 1 μl of the bacterial suspension was used as a template for multiplex PCR detection.

[0103] 2. PCR reaction: the reaction conditions and reaction system are the same as in Example 1.

[0104] 3. Test results and description

[0105] Analyzed by 1% agarose gel electrophoresis. The results showed that the corresponding specific bands were amplified in the samples added with bacteria (see the results in the attached Figure 5 6th track), and no corresponding bands appeared in the samples without artificial addition of bacteria (see attached Figure 5 track 7).

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Abstract

The invention relates to a method of detecting Francisella tularensis by a multiple PCR technique, belonging to the technical field of detecting biological agent. The invention comprises steps of: using the related genes lpxF, lpxE and flmK of the endotoxin of the Francisella tularensis as the specificity targets, carrying out comparison by complete sequence Blastp of NCBI microbial genome to determine the specificities of the three genes, designing the related primer and conducting detection by the multiple PCR technique; after three strips, which are 447bp,717bp and 1004bp in turn, appear in the electrophoresis detection, judging the strips to be masculine according to the appearance of three specificity strips, otherwise, judging the strips to be feminine. The invention has the advantages of simple and convenient operation, easy promotion and application, short detecting cycle, high sensitivity, good specificity and higher detecting rate, wherein, with respect to 5 to 7 days neededto obtain the result by the traditional detecting method, the detecting cycle of the method, which is 3 hours, is much shorter than that of the traditional method, thus greatly improving the detecting efficiency; moreover, the detecting limit, which is 0.01ng / [mu]L and 5X10<5>cfu / mL, reaches or exceeds that of the traditional detecting method.

Description

technical field [0001] The invention relates to a method for detecting Francisella tularensis, in particular to a method for rapidly detecting Francisella tularensis through multiple PCR. The invention belongs to the technical field of biological warfare agent detection. Background technique [0002] Microorganisms are tiny and closely related to human life. Microorganisms are "everywhere and everywhere" in nature, covering many beneficial and harmful species, and widely involving many fields such as health, medicine, industry and agriculture, and environmental protection. Infectious diseases caused by microorganisms have rapid onset, high mortality, rapid spread, and wide spread, which not only seriously endanger people's health but also easily cause public psychological panic. Therefore, microorganisms are often chosen by terrorists to create bioterrorism incidents Preferred target. With the rapid development of modern biotechnology, it is becoming more and more difficu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04
Inventor 王小元李颜颜许阳光李烨史峰吝易
Owner JIANGNAN UNIV
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