Fluorescent quantitative PCR detection primer group and kit for enterocytozoon hepatopenaei based on TaqMan-MGB probe

A detection kit and a technology for shrimp E. hepatobacteria are applied in the field of shrimp E. hepatobacteria fluorescence quantitative PCR detection primer sets and kits, which can solve the problems such as the reduction of the resolution of the experimental results by the fluorescent background, and achieve the improvement of sensitivity and efficiency. Good stability and improved sensitivity

Pending Publication Date: 2020-09-08
GUANGXI ACADEMY OF FISHERY SCI
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0008] Compared with ordinary PCR, the detection and analysis process of fluorescent quantitative PCR is automatically completed by the machine in a closed single tube, which has the advantages of high degree of automation, effective solution to PCR product contamination, and quantitative detection of the strength of pathogen infection. The method of detecting pathogens is considered to be the most ideal method; TaqMan-MGB probe is a fluorescent probe used in fluorescent quantitative PCR, and the quencher group at its 3' end is non-luminescent MGB (Minor Groove Binder) Conjugates, which can achieve high Tm value, shorten the length of the probe and facilitate annealing with the template, which is beneficial to improve the sensitivity of the method, and the non-luminescent quenching group at the 3' end of the probe is closer to the reporter group at the 5' end in space , so that the fluorescence background is reduced and the resolution of the experimental results is higher

Method used

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  • Fluorescent quantitative PCR detection primer group and kit for enterocytozoon hepatopenaei based on TaqMan-MGB probe
  • Fluorescent quantitative PCR detection primer group and kit for enterocytozoon hepatopenaei based on TaqMan-MGB probe
  • Fluorescent quantitative PCR detection primer group and kit for enterocytozoon hepatopenaei based on TaqMan-MGB probe

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Design and synthesis of primers and TaqMan-MGB probes.

[0046] Using PrimerExpress 3.0 software, according to the design principles of fluorescent quantitative PCR primers and MGB probes, multiple sets of specific amplification primers and TaqMan-MGB probes were designed in the conserved region of the EHP-SSU rDNA gene (KF362129), and analyzed by Oligo7.0 software Comparing and appropriately revising and optimizing, 5 groups of primers and probes with ideal evaluation (see Table 1) were obtained, and the BLAST homology comparison was performed through the NCBI database to verify the specificity, and then handed over to Sangon Bioengineering (Shanghai) Co., Ltd. for synthesis, among which The 5' end of the probe is labeled with the fluorescent dye FAM, and the 3' end is labeled with the MGB group.

[0047] Table 1 Primer and probe information for primary screening

[0048]

[0049] Fluorescent quantitative PCR was performed simultaneously on two EHP DNA templates wi...

Embodiment 2

[0055] Extraction of pathogenic nucleic acid and preparation of recombinant plasmid standards.

[0056] Take 30mg of positive disease tissue, and extract common shrimp pathogens EHP, SIV, WSSV, IHHNV, Vp according to the instructions of the animal tissue genomic DNA rapid extraction kit AHPND , V.harveyi, and V.vnlnificus nucleic acid DNA, and finally add 60 μL of elution buffer to dissolve the DNA, and store at –20°C for later use.

[0057] Using EHP DNA as a template, sterilized ddH 2 O is a blank control, and the fluorescence quantitative PCR reaction is carried out. Refer to the instructions for use of reagents and instruments.

[0058] Add 2×Probe qPCR Premix Ex Taq to the 50μL reaction system TM 25 μL, 0.5 μL of ROX ReferenceDyeⅡ, 1 μL of upstream and downstream primers EHP-qF66 and EHP-qR66 (10 μmol / L), 1 μL of TaqMan-MGB probe EHP-qP66 (10 μmol / L), 5 μL of template DNA, and sterilized ddHO 2 O to make up to 50 μL. The reaction conditions were: pre-denaturation at ...

Embodiment 3

[0062] Optimization of reaction conditions for fluorescent quantitative PCR.

[0063] With 3 gradients (1.0×10 9 , 1.0×10 6 , 1.0×10 3 copies / μL) of the standard DNA as a template, the reaction system is referred to Example 2, wherein the concentration of the upstream and downstream primers is set to 0.1-0.8 μmol / L in increments of 0.1 μmol / L, and the concentration of other components remains unchanged. According to the amplification reaction The cycle threshold value (Cycle threshold value, Ct value) and the fluorescence signal intensity (ΔRn) of the amplification curve were used to select the optimal primer concentration.

[0064] The results show that, respectively, with 1.0×10 9 , 1.0×10 6 , 1.0×10 3 The copy / μL standard is the template, and the results of fluorescent quantitative PCR amplification when the upstream and downstream primer concentrations are 0.1-0.8 μmol / L show that as the primer concentration increases, ΔRn increases and the Ct value decreases, but the...

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Abstract

The invention discloses a fluorescent quantitative PCR detection primer group and kit for enterocytozoon hepatopenaei based on a TaqMan-MGB probe, and belongs to the technical field of virus detection. The detection primer group specifically comprises a primer for detecting enterocytozoon hepatopenaei (EHP) and a TaqMan-MGB probe, and the invention also provides the kit and a detection method based on the detection primer and the probe. According to the designed specific primer and TaqMan-MGB probe, a recombinant plasmid standard substance pMD18-T-SSUEHP is prepared, and the TaqMan-MGB probe fluorescent quantitative PCR method for detecting EHP is established. The detection primer group and kit have the advantages of strong specificity, high sensitivity, good repeatability, wide quantitative range, simplicity, rapidness and the like, are suitable for quantitative detection of EHP of shrimp samples, and can provide a new technical means for rapid quantitative detection and real-time monitoring of EHP.

Description

technical field [0001] The invention relates to the technical field of virus detection, and more specifically relates to a TaqMan-MGB probe-based fluorescent quantitative PCR detection primer set and kit for Shrimp Enterocystis hepatica. Background technique [0002] Enterocytozoon hepatopenaei (Enterocytozoon hepatopenaei, EHP) is an obligate intracellular parasitic microsporidia belonging to the family Enterococidae and the genus Enterococcus. It was isolated and officially named and reported that it mainly infects important cultured shrimp such as Litopenaeus vannamei and Penaeus monodon, and is one of the main pathogens that have harmed cultured shrimp in the past 10 years. EHP can be transmitted horizontally and vertically, but it is mainly infected by ingesting fresh and live bait of parasitic EHP, diseased shrimp, dead shrimp or sporozoites in water. EHP infection mainly leads to slow or even stagnant growth of cultured prawns, manifested as emaciation, uneven size, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6893C12Q1/686C12N15/11
CPCC12Q1/6893C12Q1/686C12Q2563/107C12Q2545/114C12Q2561/101
Inventor 童桂香韦信贤林勇谭红连陈秀荔熊建华黄光华杨慧赞王瑞
Owner GUANGXI ACADEMY OF FISHERY SCI
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