Fluorogenic quantitative PCR detecting method of vibrios

A fluorescence quantitative and detection method technology, applied in the field of microbial detection, can solve the problems of insufficient accuracy, detection specificity, limited sensitivity, and low detection efficiency, and achieve the effects of high sensitivity, convenient, fast and accurate detection, and strong specificity.

Inactive Publication Date: 2015-10-28
YANGTZE UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] PCR detection method is one of the effective methods to detect Vibrio, but due to the limited detection specificity and sensitivity of current PCR detection, when Vibrio content is low, it cannot be detected in time, resulting in low detection efficiency and insufficient accuracy

Method used

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  • Fluorogenic quantitative PCR detecting method of vibrios
  • Fluorogenic quantitative PCR detecting method of vibrios
  • Fluorogenic quantitative PCR detecting method of vibrios

Examples

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Embodiment 1

[0044] Example 1: Fluorescent quantitative PCR detection of Vibrio using toxR gene primers

[0045] This embodiment adopts the toxR gene primer to detect Vibrio according to the following main steps, and further tests the sensitivity:

[0046] (1) Preparation of bacteria-specific primers

[0047] The toxR gene toxR of Vibrio riverina was downloaded from NCBI, and the primer sequence was designed by Primer Premier 5.0. The sequence of the toxR gene primer pair is shown in Table 1.

[0048] Table 1 toxR gene primer pair

[0049]

[0050] (2) Bacterial culture and template acquisition

[0051] According to the requirements of aseptic operation, pick and inoculate a single colony of Vibrio riverina into 5mL LB liquid medium, place it in an incubator at 30°C and 200rpm, and culture it with shaking for 20h until the bacterial liquid is turbid. Take 20μL and spread it on the LB agar plate placed in an incubator at 30°C for 24 hours. Observe the growth of the above strains, selec...

Embodiment 2

[0058] Example 2: Fluorescent quantitative PCR detection of Vibrio using flaA gene primers

[0059] This embodiment adopts the flaA gene primer pair to detect Vibrio according to the following main steps, and further tests the sensitivity:

[0060] (1) Preparation of bacteria-specific primers

[0061] The flagellin flaA gene of Vibrio anguillarum was downloaded from NCBI, and the primer sequence was designed by Primer Premier5.0. The primer pair sequence of flaA gene is shown in Table 2.

[0062] Table 2 flaA gene primer pair

[0063]

[0064] (2) Bacterial culture and template acquisition

[0065] According to the requirements of aseptic operation, pick and inoculate a single colony of Vibrio anguillarum into 5mL LB liquid medium, place it in an incubator at 35°C and 200rpm, and culture it with shaking for 15h, until the bacterial liquid is cloudy, take 20μL and spread it on the LB agar plate placed in an incubator at 35°C for 20 h. Observe the growth of the above stra...

Embodiment 3

[0072] Example 3: Fluorescent quantitative PCR detection of Vibrio using pyrH gene primers

[0073] This embodiment adopts the pyrH gene primer pair to detect Vibrio according to the following main steps, and further tests the sensitivity:

[0074] (1) Preparation of bacteria-specific primers

[0075] The pyrH gene of Vibrio alginolyticus was downloaded from NCBI, and the primer sequence was designed by Primer Premier 5.0. The primer pair sequence of pyrH gene is shown in Table 3.

[0076] Table 3 pyrH gene primer pair

[0077]

[0078] (2) Bacterial culture and template acquisition

[0079] According to the requirements of aseptic operation, pick and inoculate a single colony of Vibrio alginolyticus into 5mL LB liquid medium, place it in an incubator at 35°C and 200rpm, and culture it with shaking for 15h, until the bacterial liquid is cloudy, take 20μL and spread it on LB agar Plates were placed in an incubator at 35°C for 20 hours. Observe the growth of the above str...

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Abstract

The invention provides a fluorogenic quantitative PCR detecting method of vibrios. The method comprises the steps that DNA of bacterial colonies obtained from culturing sample bacteria is extracted, fluorogenic PCR amplification is carried out on the DNA of the bacterial colonies through specific primers, and the specific primers are one of toxR gene primer pairs indicated by a sequence table SEQ ID No.1-2, flaA gene primer pairs indicated by a sequence table SEQ ID No.3-4, and pyrH gene primer pairs indicated by a sequence table SEQ ID No.5-6. The method is high in specificity and sensitivity, the pathogenic bacterium variety can be further determined as vibrio fluvialis or vibrio anguillarum or vibrio alginolyticus, the minimum concentration capable of being detected is 1.0*10<-6> nmol/L, 1.0*10<-7> nmol/L and 1.0*10<-8> nmol/L, detecting is convenient, fast and accurate, and great significance in preventing vibriosis, one of main bacterial diseases of maricultural animals, is achieved.

Description

technical field [0001] The invention belongs to the technical field of microbial detection, in particular to a fluorescent quantitative PCR detection method for Vibrio. Background technique [0002] Vibrio is a kind of halophilic Gram-negative bacilli widely distributed in the marine environment. Now there are more than 10 kinds of pathogenic bacteria in aquaculture, including Vibrio riverina, Vibrio eel, and Vibrio alginolyticus. Vibrio riverina, Vibrio anguillarum and Vibrio alginolyticus are opportunistic pathogenic bacteria, which can induce diseases when aquaculture animals encounter adverse stimuli or injuries under adverse environmental conditions. [0003] With the rapid development of artificial breeding and the ecological changes of the breeding waters, vibriosis has become one of the main bacterial diseases of marine animals. It has the characteristics of wide prevalence, high morbidity, great harm, and high mortality, which has caused a huge impact on the culti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/689C12Q2561/101C12Q2545/114C12Q2531/113Y02A50/30
Inventor 许巧情罗凯郜卫华张平英朱大世李升康温小波
Owner YANGTZE UNIVERSITY
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