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Fluorescent quantitative PCR method for detecting vibrio parahaemolyticus causing acute hepatic pancreatic necrosis

A technology of vibrio hemolyticus and fluorescence quantification, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, and microorganism measurement/inspection, which can solve the problems of difficult detection, poor sensitivity, and long detection time of latent infection samples, etc. problem, to achieve the effect of improved stability, good quenching effect and small background

Pending Publication Date: 2019-08-09
GUANGXI ACADEMY OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] However, in actual detection work, one-step PCR is not sensitive enough, and it is difficult to detect latently infected samples; nested PCR is highly sensitive, but requires two rounds of PCR and electrophoresis, which is cumbersome to operate and takes a long time to detect; LAMP is sensitive and fast, but easy False positives occur; TaqMan probe fluorescence quantitative PCR is not sensitive due to the length of the probe is too long

Method used

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  • Fluorescent quantitative PCR method for detecting vibrio parahaemolyticus causing acute hepatic pancreatic necrosis
  • Fluorescent quantitative PCR method for detecting vibrio parahaemolyticus causing acute hepatic pancreatic necrosis
  • Fluorescent quantitative PCR method for detecting vibrio parahaemolyticus causing acute hepatic pancreatic necrosis

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Experimental program
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Effect test

Embodiment 1

[0053] Real-time quantitative PCR method for detection of Vibrio parahaemolyticus causing acute hepatopancreatic necrosis

[0054] 1. Design and synthesis of primers and TaqMan-MGB probes

[0055] with Vp AHPND PirA Vp The gene is the detection target gene, targeting PirA Vp The gene sequence uses PrimerExpress3.0 software to design specific primers and TaqMan-MGB probes, the target product length is 60bp, the 5' end of the probe is labeled with the fluorescent group FAM, and the 3' end is labeled with the MGB group, primers and probe sequences for:

[0056] upstream primer PirA Vp -qF: 5'-CAAACGGAGGCGTCACAGA-3',

[0057] downstream primer PirA Vp -qR: 5′-GACCGACTTCCGGGATGAT-3′,

[0058] ProbePirA Vp -P: 5'-FAM-AGACAGCAAACATACACC-MGB-3'.

[0059] 2. DNA extraction and preparation of recombinant plasmid standards

[0060] (1)Vp AHPND Recovery and DNA extraction:

[0061] Take the frozen-thawed strain and inoculate it on TSA medium, incubate at 36±1°C for 18-24h, the...

Embodiment 2

[0083] sensitivity test

[0084] With 9 gradients (1.0×10 1 -1.0×10 9 copy / μL) standard DNA as a template, and use the optimized fluorescent quantitative PCR method to detect; use the data analysis software that comes with the instrument to analyze, and establish the logarithm and CT value (Y) of the initial template copy number (X) The standard curve and standard equation were used, and the quantitative range and sensitivity of the method were judged according to the amplification results of each standard DNA. Test results such as figure 2 (Standard (1.0×10 1 -1.0×10 9 copy / μL) fluorescence quantitative PCR amplification curve) and image 3 (Fluorescence quantitative PCR detection of Vp AHPND standard curve) shown.

[0085] Depend on figure 2 It can be seen that the 9 gradients (1.0×10 1 -1.0×10 9 copies / μL) of the standard DNA was amplified, and the results showed that: high-concentration standard (1.0×10 3 -1.0×10 9 copies / μL) of the amplification curves all s...

Embodiment 3

[0090] specificity test

[0091] with Vp AHPND , Vibrio parahaemolyticus, Vibrio harveyi, Vibrio alginolyticus, Vibrio vulnificus, Vibrio riverine, WSSV, IHHNV, EHP and SHIV nucleic acid samples were used as templates, and the optimized fluorescent quantitative PCR method was used for detection and evaluation The specificity of the method, test results such as Figure 4 (Vp AHPND Specific detection results of fluorescent quantitative PCR).

[0092] Among them, Vibrio bacteria (Vp AHPND -BH170612 strain, Vibrio parahaemolyticus VP-FCG151118, Vibrio harveyi VH-QZ160809, Vibrio alginolyticus VA-QZ160809, Vibrio vulnificus VV-BH160905, Vibrio riverina VF-FCG170910) recovery and DNA extraction methods are : Streak inoculation of frozen and thawed Vibrio strains on TSA medium, culture at (36±1)°C for 18-24h, then select a single colony and inoculate on TSB medium, and continue to cultivate at (36±1)°C for 18-24h . Take 1.0-3.0 mL of the above bacterial solution, centrifuge at ...

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Abstract

The invention discloses a fluorescent quantitative PCR method for detecting vibrio parahaemolyticus causing acute hepatic pancreatic necrosis. The method specifically comprises the following steps of(1) taking the PirAVp gene of VpAHPND as a detection target gene and designing a specific primer and a TaqMan-MGB probe; (2) extracting the DNA of the VpAHPND, constructing a recombinant plasmid, preparing a standard substance, and storing at -20 DEG C for use; (3) performing a quantitative PCR, and establishing a standard curve and a standard equation between the logarithm of the initial templatecopy number and the threshold cycle number; (4) detecting the to-be-detected sample with the primer and the probe in the step (1),and based on the measured threshold cycle number, according to the standard curve, calculating the copy number of the VpAHPND in the to-be-detected sample. The Fluorescent quantitative PCR method for detecting the VpAHPND for non-diagnostic purposes established by theinvention has the advantages of high sensitivity, strong specificity, good reproducibility, rapid quantification and the like, and can be used for clinically detecting the VpAHPND of shrimp samples and water samples.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a fluorescent quantitative PCR method for detecting acute hepatopancreatic necrosis vibrio parahaemolyticus. Background technique [0002] Since 2009, Acute hepatopancreas necrosis disease (AHPND) has been widespread in shrimp farming worldwide, mainly harming Litopenaeus vannamei, Penaeus monodon and Fenneropenaeus chinensis), resulting in a sharp drop in the production of prawns, which has brought huge economic losses to the shrimp farming industry. The prawn farming industry in my country, which is dominated by Litopenaeus vannamei farming, has also suffered heavy losses. The cause of the disease has no obvious symptoms and no obvious precursors before death. Most of the prawns die at the bottom of the pond and are not easy to be found. It is commonly known as "stealing death disease". Since the disease mainly occurs 7-30 days after the shrimps are stocked, so Als...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/06C12R1/63
CPCC12Q1/689C12Q1/6851C12Q2561/101C12Q2545/114C12Q2531/113
Inventor 韦信贤童桂香吴祥庆杨琼黄国秋熊建华谭红连陈静
Owner GUANGXI ACADEMY OF FISHERY SCI
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