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Primer and method for fluorescent quantitative PCR detection of Enterocytozoon hepatopenaei (EHP)

A fluorescence quantitative, Enterospora hepatis technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of time-consuming, cumbersome operation, etc. The effect of shortening the detection time

Pending Publication Date: 2021-10-19
杭州市农业技术推广中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the nested-PCR detection technology with SWP as the target gene has been established, but this technology can only be used for qualitative detection, and the operation is cumbersome, requiring multiple pairs of primers for multiple amplification reactions, which takes a long time

Method used

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  • Primer and method for fluorescent quantitative PCR detection of Enterocytozoon hepatopenaei (EHP)
  • Primer and method for fluorescent quantitative PCR detection of Enterocytozoon hepatopenaei (EHP)
  • Primer and method for fluorescent quantitative PCR detection of Enterocytozoon hepatopenaei (EHP)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment example 1

[0035] The pMD19T cloning vector involved in this implementation case, DH5α cloning competence and easydilution for constructing standards for diluted plasmids were purchased from Takara Biotechnology Co., Ltd.

[0036] The EHPSWP sequence information involved in this implementation case has been published in NCBI.

[0037] This implementation case constructs the EHP-SWP standard plasmid, the main steps are as follows:

[0038] 1) Extract the total DNA of the hepatopancreas of Penaeus vannamei naturally infected with EHP;

[0039] 2) Design primers according to the EHP-SWP sequence published by Ncbi, and obtain the EHP-SWPORF sequence by PCR amplification;

[0040] 3) connect the pMD 19T plasmid, and transform DH5α;

[0041] 4) Positive clones were obtained through sequencing identification, and after expansion and cultivation, plasmids were extracted;

[0042] 5) The Nanodrop nucleic acid analyzer measures the plasmid concentration, and converts the EHP infection copy numb...

Embodiment example 2

[0045] Screening of implementation case 2 specific primers

[0046] On the basis of the preliminary screening, the following 2 pairs of primers were finally determined for multiple comparison experiments:

[0047] Primer pair 1EHP146F: TGGCGGCACAATTCTCAA

[0048] EHP146R: GCTGTTTGTCTCCAACTGTA

[0049] Primer pair 2F: TGGCGGCACAATTCTCAAACAT

[0050] R: GCTGTGTCTGTGTAAATATCGTCTC

[0051] In this implementation case, TB Green Premix Ex Taq II (TliRNaseH Plus) (2*) was purchased from Takara Biotechnology Co., Ltd.

[0052] In this implementation case, by constructing a standard curve, the amount of EHP infection in Penaeus vannamei juveniles (<2cm) and "growth retarded" adult Penaeus vannamei was detected. The specific steps are as follows:

[0053] 1. Sample collection and DNA extraction: take whole individuals from larvae, larvae and juveniles smaller than 2cm, and hepatopancreas from juveniles larger than 2cm, subadult shrimp and adults. Samples were collected in the range...

Embodiment example 3

[0063] In this implementation case, TB Green Premix Ex Taq II (TliRNaseH Plus) (2*) was purchased from Takara Biotechnology Co., Ltd.

[0064] In this implementation case, by constructing a standard curve, the amount of EHP infection in Penaeus vannamei juveniles (<2cm) and "growth retarded" adult Penaeus vannamei was detected. The specific steps are as follows:

[0065] 1. Sample collection and DNA extraction: take whole individuals from larvae, larvae and juveniles smaller than 2cm, and hepatopancreas from juveniles larger than 2cm, subadult shrimp and adults. Samples were collected in the range of 10-50 mg, and the total DNA of the samples was extracted using the Qiagen DNA Mini Kit kit in strict accordance with the kit instructions.

[0066] 2. Fluorescent quantitative PCR detection:

[0067] 1) The reaction system is as follows, with a total volume of 25ul: TB Green Premix Ex Taq II (TliRNaseH Plus) (2X): 12.5ul, 1.25ul each of the primers of the sequences described in E...

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Abstract

The invention relates to a method for fluorescent quantitative PCR detection of Enterocytozoon hepatopenaei (EHP). According to the method, a pair of specific primers taking EHP spore wall protein SWP as a target gene and an SWP standard substance kit are used. The standard substance kit comprises an SWP standard plasmid diluted by 10 times of gradient and a bacterial suspension of an SWP standard strain. A TBGreen fluorescent dye method is adopted, the EHP infection of cultured prawns is quantitatively detected by detecting the EHP SWP, high sensitivity and specificity are achieved, the method can be used for rapid detection and diagnosis of EHP, and a foundation is laid for monitoring and early prevention and control of EHP infection.

Description

technical field [0001] The invention relates to a rapid detection method of prawn enterospora hepatica by fluorescence quantitative PCR, which belongs to the technical field of aquatic organism pathogen detection. Background technique [0002] Enterocytozoon hepatopenaei (EHP) is a type of obligate intracellular parasitic microsporidia, which mainly infects the intestinal epidermis and hepatopancreas of cultured prawns, and parasitizes the hepatopancreas tubular epithelial cells. The infection leads to a decline in the digestion and absorption function of the prawns, which affects the absorption of nutrients. There are no obvious symptoms and death in the early stage of the infection, and it is difficult to find out. Usually, after 30-45 days of cultivation, it can be seen that the cultured prawns grow slowly and vary in size. And there was a significant negative correlation between the individual growth rate of shrimp and the EHP loading index in the body. In addition, EHP...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6893C12Q1/6851C12Q1/06C12N15/11
CPCC12Q1/6893C12Q1/6851C12Q2600/166C12Q2531/113C12Q2545/114C12Q2563/107
Inventor 叶键许婷施礼科姜路辛陈凡郭水荣王力蒋静陈喆
Owner 杭州市农业技术推广中心
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