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Multiple PCR detection primers for simultaneously detecting HPV, MBV and IHHNV of prawns, kit and application

A detection kit and shrimp technology, applied in the direction of DNA/RNA fragments, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems affecting the sensitivity and accuracy of detection results, and achieve low detection limit and good specificity , the effect of high sensitivity

Active Publication Date: 2016-02-10
THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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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  • Multiple PCR detection primers for simultaneously detecting HPV, MBV and IHHNV of prawns, kit and application
  • Multiple PCR detection primers for simultaneously detecting HPV, MBV and IHHNV of prawns, kit and application
  • Multiple PCR detection primers for simultaneously detecting HPV, MBV and IHHNV of prawns, kit and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1: Tests for detection of prawn hepatopancreatic parvovirus, baculovirus of Penaeus monodon, infectious subcutaneous and hematopoietic tissue necrosis virus

[0057] 1) Reagents

[0058] 10×TaqBuffer: Tris-HCl100mM, KCl500mM, MgCl 2 20mM, dNTPs 2mM, Taq enzyme 1U / μL;

[0059] Primers: HPV-F, HPV-R, MBV-F, MBV-R, IHHNV-F, IHHNV-R 10μM each, pure water, positive quality control DNA 10μg / ml;

[0060] According to the nucleic acid sequences of Hepatopancreatic parvovirus (HPV), Monodon baculovirus (MBV), Infectious hypodermal and hematopoietic necrosis virus (IHHNV), the present invention is designed to detect hepatopancreas Specific primers for parvovirus, Penaeus monodon baculovirus, infectious hypodermic and hematopoietic necrosis virus. In the present invention, through experimental screening of the designed primers, a group of primers with very high sensitivity and specificity for hepatopancreatic parvovirus, Penaeus monodon baculovirus, infectious subcuta...

Embodiment 2

[0093] Example 2: Simultaneous detection of prawn hepatopancreatic parvovirus, Penaeus monodon baculovirus, infectious subcutaneous and hematopoietic tissue necrosis virus Multiplex PCR detection primers, kits and detection methods for further effect detection.

[0094] 1. Sensitivity experiment

[0095] Dilute the above-mentioned artificially synthesized positive quality control substance, and successively dilute to 1×10 10 , 1×10 9 , 1×10 8 , 1×10 7 , 1×10 6 , 1×10 5 , 1×10 4 , 1×10 3 , 1×10 2 , 1×10 1 copies / μL, which were used as multiplex PCR templates for sensitivity experiments. see results Figure 2-5 .

[0096] figure 1 It is the result diagram of the sensitivity experiment for the detection of prawn hepatopancreas parvovirus in the multiplex PCR system. Where M is TakaraDL2000DNAMarker, 1-10 is positive standard (from 1 to 10 are 1×1010, 1×109, 1×108, 1×107, 1×106, 1×105, 1×104, 1×103 , 1×102, 1×101 copy / μL), 11 is the negative control. It can be se...

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Abstract

The invention discloses multiple PCR detection primers for simultaneously detecting HPV, MBV and IHHNV of prawns, a kit and application. The primers are respectively HPV-F and HPV-R, MBV-F, MBV-R, IHHNV-F and IHHNV-R, sequences are shown as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. The primers do not cross mutually, do not cross with other viruses, are low in false positive rate, can specifically detect out three different types of virus strains distributed around the world, has wide applicability, very high specificity and sensitivity and is low in detection limit. In addition, detected virus gene segments are not needed to undergo cloning, sequencing and sequence alignment. Whether a sample to be tested contains one, two or three of the three types of viruses or not can be detected out through one experiment.

Description

technical field [0001] The present invention relates to marine biological pathogen detection technology, in particular to a simultaneous detection of shrimp hepatopancreatic parvovirus (HPV), monodon baculovirus (Monodonbaculovirus, MBV), infectious subcutaneous and hematopoietic necrosis virus (Infectioushypodermalandhematopoieticnecrosisvirus, IHHNV) ) for multiplex PCR detection primers, kits and uses. Background technique [0002] Shrimp hepatopancreatic parvovirus (Hepatopancreaticparvovirus, HPV) belongs to Parvoviridae, Densoviridae subfamily, with an average diameter of 22-23nm, inclusion body, single-stranded DNA virus (ssDNA), non-enveloped icosahedron. It is a chronic infection type in virology, with strong infectivity and high incidence. After shrimps are infected with hepatopancreatic parvovirus, the epithelial cells of the hepatopancreas are enlarged and degenerated, especially in the fully developed stage of the disease. As a result, the diseased shrimp lose...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68C12Q1/70
Inventor 陈明谅刘国胜陈建明杨慧
Owner THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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