Duplex PCR (polymerase chain reaction) detection method and kit for simultaneously detecting shrimp enterocytozoon hepatopenaei and tetrapod iridovirus 1

A technique for shrimp hepatic Enterocystis and iridescent virus, which is applied in the field of double PCR detection methods and kits, can solve the problems affecting the sensitivity and accuracy of detection results, and achieve the effects of low detection limit, high sensitivity and wide application prospects

Pending Publication Date: 2022-07-08
日照市海洋与渔业研究院(日照市海域使用动态监视监测中心日照市水生野生动物救护站)
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  • Abstract
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Problems solved by technology

However, multiplex PCR detection is also affected by many factors, such as the activity of DNA polymerase, the quality of the template, the specificity of the designed viral primers, PCR reaction conditions, and the competition between different virus samples for reagents, etc. will affect the detection results. sensitivity and accuracy

Method used

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  • Duplex PCR (polymerase chain reaction) detection method and kit for simultaneously detecting shrimp enterocytozoon hepatopenaei and tetrapod iridovirus 1
  • Duplex PCR (polymerase chain reaction) detection method and kit for simultaneously detecting shrimp enterocytozoon hepatopenaei and tetrapod iridovirus 1
  • Duplex PCR (polymerase chain reaction) detection method and kit for simultaneously detecting shrimp enterocytozoon hepatopenaei and tetrapod iridovirus 1

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1 Detects shrimp Enteroplasma hepatis, Decapod iridescent virus 1 test

[0044] (1) Required reagents:

[0045] 10×TaqBuffer: Tris-HCl 100mM, KCl 500mM, MgCl2 20mM, dNTPs 2mM, Taq enzyme 1U / μL;

[0046] (2) Primers required:

[0047] EHP-F, EHP-R, DIV1-F, DIV1-R each 10μM, pure water, positive quality control substance (positive control group) DNA 10μg / ml;

[0048] According to the nucleic acid sequences of Enterocytozoon hepatopenaei (EHP) and Decapod iridescent virus 1 (Decapod iridescent virus 1), the present invention designs specific primers for detecting Enterocytozoon hepatopenaei and Decapod iridescent virus 1 respectively. In the present invention, the designed primers are screened experimentally to screen out a group of primers with very high sensitivity and specificity for both shrimp Enteroplasma hepatis and decapod iridovirus 1, and the primer sequences are as follows:

[0049] Enterobacter hepatis

[0050] Upstream primer: EHP-F: GAGAGTAGCGG...

Embodiment 2

[0071] Embodiment 2: Double PCR detection primers, kits and detection methods for simultaneous detection of Enterocytoplasma hepatis and Decapoda iridescent virus 1 are used for further effect detection.

[0072] A sensitivity test

[0073] Dilute the above-mentioned synthetic positive quality control products, and dilute them in sequence to 1×108, 1×107, 1×106, 1×105, 1×104, 1×103, 1×102, 1×101 copies / μL, Sensitivity experiments were performed as double PCR templates, respectively. see the results Figure 1-3 .

[0074] figure 1 This is the graph of the sensitivity test results for the detection of Enterobacter hepatis in the multiplex PCR system. M is TakaraDL2000 DNA Marker, 1-8 are positive standards (from 1 to 8 are 1×108, 1×107, 1×106, 1×105, 1×104, 1×103, 1×102, 1 ×101 copies / μL), 9 is the negative control. As can be seen from the figure, lanes 1-8 all have clear PCR amplification bands of about 512 bp, and the detection sensitivity of shrimp E. hepatobacteria is ...

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Abstract

The invention belongs to the technical field of marine organism pathogen detection, and particularly relates to a dual PCR (polymerase chain reaction) detection method and kit for simultaneously detecting shrimp enterocytozoon hepatopenaei and decapod iridovirus 1. The invention provides a duplex PCR (polymerase chain reaction) detection method for simultaneously detecting enterocytozoon hepatopenaei and tetrapod iridovirus 1, used PCR primers are shown as SEQ ID NO: 1 and SEQ ID NO: 2, and detected pathogenic gene segments do not need to be cloned, sequenced and subjected to sequence alignment. Whether a sample to be detected contains one or two of the two pathogens or not can be detected through one experiment. And a plurality of limitations of complicated operation, low sensitivity, low applicability and the like of the conventional single detection are overcome.

Description

technical field [0001] The invention belongs to the technical field of marine biological pathogen detection, and in particular relates to a double PCR detection method and a kit for simultaneously detecting shrimp Enteroplasma hepatis and Decapod iridovirus 1. Background technique [0002] Enterocytozoon hepatopenaei (EHP) and Decapod iridescent virus 1 (DIV1) are two major diseases that seriously endanger the health of shrimp farming. Critical diseases of crustaceans to which it is to be notified. [0003] At present, the diagnostic methods of shrimp virus disease mainly include pathological and biological methods, immunodiagnosis, molecular hybridization and PCR. The established conventional PCR technology can only detect one pathogen at a time, which is time-consuming. The multiplex PCR technology is a special PCR method. Multiple pairs of virus-specific primers are added to a reaction system, which can detect multiple pathogens at the same time, thereby shortening the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6893C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/6893C12Q1/686C12Q2600/16C12Q2600/166C12Q2537/143C12Q2545/113Y02A50/30
Inventor 陈晓玲王君霞季本安许传堂韩涛于为民于盟盟
Owner 日照市海洋与渔业研究院(日照市海域使用动态监视监测中心日照市水生野生动物救护站)
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