Fluorescent quantitative PCR (polymerase chain reaction) method for detecting enterocytozoon hepatopenaei of prawns and corresponding kit

A fluorescence quantification and kit technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of low detection sensitivity, long detection time, and high false positives, and achieve high sensitivity, convenient operation, and rigorous results. and accurate effect

Pending Publication Date: 2020-08-04
广东美格基因科技有限公司
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The first three methods are complicated to operate and take a long time, and generally need to be confirmed in a molecular laboratory, and their detection sensitivity is low, so they can only diagnose the obvious prawns. It cannot be quantified, which to a certain extent limits the popularization and application of common PCR detection methods in production; the loop-mediated isothermal amplification method, although the detection time is fast and the operation is simple, has high false positives and has been criticized by professionals

Method used

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  • Fluorescent quantitative PCR (polymerase chain reaction) method for detecting enterocytozoon hepatopenaei of prawns and corresponding kit
  • Fluorescent quantitative PCR (polymerase chain reaction) method for detecting enterocytozoon hepatopenaei of prawns and corresponding kit
  • Fluorescent quantitative PCR (polymerase chain reaction) method for detecting enterocytozoon hepatopenaei of prawns and corresponding kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Embodiment 1 detection primer amplification standard curve

[0059] 1. Microbial culture

[0060] Use LB medium as the culture medium for the E. hepatica plasmid of prawns, and it grows well in the plate with pH 7.0~7.4. The diameter of the colony on the plate is 2mm, round, smooth and transparent. The vigorously growing microbial samples were selected for subsequent experiments.

[0061] 2. Genomic DNA Extraction

[0062] Genomic DNA was extracted according to the instructions of Qiagen's QIAamp DNA Mini Kit.

[0063] 3. Production of standard plasmids for amplified products

[0064] Using the DNA extracted from prawn Enterospora hepatica, amplified with the primers of 18SrRNA gene, β-tubulin gene and SWP1 gene respectively, after adding A treatment, the amplified product was ligated into the T vector using T4 ligase, and passed After transduction of competent cells, the plasmid was amplified on a large scale. The primer sequences of the three genes are shown in ...

Embodiment 2

[0083] Example 2 Plasmid Repeatability Experiment

[0084] 1. Microbial culture

[0085] With embodiment 1.

[0086] 2. Genomic DNA Extraction

[0087] With embodiment 1.

[0088] 2. qPCR detection of 3 target genes

[0089] Carry out qPCR amplification of the sample according to the instructions of TaKaRa Taq PCR Mix, and obtain the Ct value reading corresponding to each pair of primers. The qPCR reaction in this experiment was set at two concentrations of 1×10 6 fg / μL and 1×10 5 fg / μL, 4 biological replicates and 5 technical replicates were set up for each concentration, and the qPCR reaction system and amplification reaction conditions are shown in Table 7.

[0090] Table 7

[0091]

[0092] 4. Result Analysis

[0093] In this example, the correlation coefficient and reliability of the amplification curves of the three genes were analyzed, as well as the amplification efficiencies at two concentrations. The measured CT values ​​were all less than 35 cycles, and a...

Embodiment 3

[0101] Embodiment 3 Positive and negative sample detection situation

[0102] 1. Microbial culture

[0103] With embodiment 1.

[0104] 2. Genomic DNA Extraction

[0105] With embodiment 1.

[0106] 3. qPCR detection of 3 target genes

[0107] Carry out qPCR amplification of the sample according to the instructions of TaKaRa Taq PCR Mix, and obtain the Ct value reading corresponding to each pair of primers. Each qPCR reaction was set up with 3 biological repetitions and 3 technical repetitions, and the qPCR reaction system and amplification reaction conditions were the same as in Example 2.

[0108] 4. Result Analysis

[0109] as shown in Table 11 and Figure 10As shown, the qPCR amplification results of 3 pairs of primers showed that E. hepatica had three positive results of 18SrRNA, β-tubulin gene and SWP1 gene, while other viruses or bacterial genera showed negative results. Therefore, for E. hepatica and other viruses or fungi, the detection of 18SrRNA, β-tubulin ge...

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Abstract

The invention discloses a fluorescent quantitative PCR (Polymerase Chain Reaction) method for detecting enterocytozoon hepatopenaei of prawns and a corresponding kit. Specific gene detection is ingeniously applied to distinguish prawn enterocytozoon hepatopenaei from other bacterial genera, and accurate bacterial genus information is obtained through comprehensive determination. Compared with an existing mainstream detection kit, the kit for detecting prawn enterocytozoon hepatopenaei has the advantages of being high in sensitivity, rapid, convenient to use, good in specificity, rigorous and accurate in judgment and the like, and has good application prospects and market value.

Description

technical field [0001] The invention belongs to the technical field of molecular detection, and in particular relates to a method for performing fluorescence quantitative PCR detection on Enterospora prawns and shrimps through specific genes and a corresponding detection kit. Background technique [0002] Microsporidium Microsporidium is a nucleated, unicellular obligate intracellular parasite found in the host range from invertebrates to vertebrates, Microsporidium has tiny spores containing a single pole silk and infectious sporoplasm. After the spores are swallowed by the host, the parasite enters the intestinal epithelium and reaches specific tissues through the blood or body cavity. [0003] Enterocytozoon hepatopenaei (EHP) is a parasitic disease caused by "Microsporidium". EHP is a new disease that appeared in the cultured prawns in my country in recent years. In 2009, a new parasite was isolated and named in the cultured Thai monodon prawn (Penaeus monodon), and t...

Claims

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Application Information

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IPC IPC(8): C12Q1/6851C12Q1/6893C12Q1/04
CPCC12Q1/6851C12Q1/6893C12Q2531/113C12Q2561/101
Inventor 唐伟束文圣杨荣束浩然黄斌蒋华
Owner 广东美格基因科技有限公司
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