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A high-sensitivity oshv-1 real-time fluorescence quantitative PCR detection kit and method

A kit and fluorescent probe technology, applied in the field of biological detection, can solve the problem that the specificity is not as good as the nest PCR technology, and achieve the effects of low cross-hybridization interference, improved specificity and high detection accuracy

Inactive Publication Date: 2018-02-13
BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this method has relatively high sensitivity, its specificity is far inferior to the nested PCR technique.

Method used

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  • A high-sensitivity oshv-1 real-time fluorescence quantitative PCR detection kit and method
  • A high-sensitivity oshv-1 real-time fluorescence quantitative PCR detection kit and method
  • A high-sensitivity oshv-1 real-time fluorescence quantitative PCR detection kit and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Embodiment 1 A kind of kit for detecting oyster herpes virus OsHV-1

[0060] The kit includes:

[0061] (1) Primer OsHV-F1, the nucleotide sequence of which is shown in SEQ ID NO: 1;

[0062] (2) Primer OsHV-R1, the nucleotide sequence of which is shown in SEQ ID NO: 2;

[0063] (3) Primer OsHV-F2, the nucleotide sequence of which is shown in SEQ ID NO: 3;

[0064] (4) primer OsHV-R2, the nucleotide sequence of which is shown in SEQ ID NO: 4;

[0065] (5) Fluorescent probe OsHV-P, the nucleotide sequence of which is shown in SEQ ID NO: 5, the 5' end of the nucleotide sequence is connected to the fluorescent group FAM, and the 3' end is connected to the quenching group BHQ1;

[0066] (6) Positive quality control product: a plasmid containing a DNA fragment amplified by using the genomic DNA of oyster herpes virus OsHV-1 as a PCR template and using primers OsHV-F1 and OsHV-R1 as PCR primers, and the concentration of the plasmid is 1.0×10 8 copy / μL;

[0067] (7) Negat...

Embodiment 2

[0068] Embodiment 2 A kind of method for detecting oyster herpes virus OsHV-1

[0069] The detection kit used is the kit described in Embodiment 1 of the present invention.

[0070] Samples to be tested: 30 oyster tissues from different sources that are known to be infected with OsHV-1 and water bodies where oysters are cultured, sample numbers 1-30, samples collected from Ningbo Fishery Products Quality Inspection Station.

[0071] Detection method:

[0072] (1) Extract viral DNA from the samples to be tested: use the viral genomic DNA / RNA extraction kit (purchased from Tiangen Biochemical Technology Co., Ltd., article number is DP315) to extract viral genomic DNA from 30 samples as the DNA to be tested. For the extraction method, please refer to the instruction manual of the extraction kit;

[0073] (2) Preparation of positive quality control products:

[0074] 1) With the genomic DNA of oyster herpes virus OsHV-1 as PCR template, carry out PCR amplification with primer O...

experiment example 1

[0111] Experimental Example 1 Sensitivity Experiment

[0112] The positive quality control product described in the present invention was diluted to 1.0×10 7 , 1.0×10 6 , 1.0×10 5 , 1.0×10 4 , 1.0×10 3 , 1.0×10 2 , 1.0×10 1 Copy / mL, as the sample to be detected, the sensitivity experiment was carried out on the detection method of Example 2, and the sensitivity was characterized by the lowest detection concentration. figure 1 For the sensitivity test results, according to figure 1 It can be seen that the plasmid copy number is 10 8 ~10 2 In the copy / mL range, there are curves with obvious exponential growth period, and the plasmid copy number is 10 2 copies / mL, there is still a fluorescent signal through amplification, so the detection sensitivity of the method provided by the invention is: 1.0×10 2 copies / mL.

[0113] The sensitivity data is as follows:

[0114] Example 2

1.0×10 2 copies / mL

Comparative example 2 public data

1.0×10 2 cop...

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Abstract

The invention provides a highly sensitive detection kit and method for OsHV-1 real-time fluorescence quantitative PCR (Polymerase Chain Reaction). The kit comprises a primer group for detecting an oyster herpes virus OsHV-1 and a fluorescence probe; and the primer group consists of four primers, and is used for a specific DNA (Deoxyribose Nucleic Acid) segment of a nested amplification oyster herpes virus OsHV-1. The detection method provided by the invention leads the four primers and the one fluorescence probe to finish nested amplification and real-time fluorescence quantitative PCR through one-step amplification in one tube, and is used for detecting the oyster herpes virus OsHV-1. The invention provides a highly sensitive, specific and accurate detection method and kit for OsHV-1 real-time fluorescence quantitative PCR, by which rapid detection can be carried out on oysters and a water body for culturing the oysters, the application is extremely convenient, based on the above-mentioned advantages, the kit is suitable for popularization and application in aquaculture units, aquaculture products supervising departments and the like, thereby having a broad application prospect.

Description

technical field [0001] The invention belongs to the technical field of biological detection and relates to the detection of oyster herpes virus OsHV-1, in particular to a real-time fluorescent quantitative PCR detection kit and method for OsHV-1. Background technique [0002] Oyster is an important cultured shellfish in the world, widely distributed in many coastal areas of my country and the world. The output of oyster farming ranks first among marine animals and has high economic value. However, large-scale death of oysters often occurs, which seriously restricts the development of oyster farming and causes serious economic losses. The oyster herpes virus OsHV-1 was first discovered in American oysters in 1972. OsHV-1 has been shown to be associated with mass mortalities in oysters. [0003] In order to reduce the economic loss caused by oyster herpes virus OsHV-1, it is often necessary to detect the aquaculture water and oysters. At present, in order to obtain relativ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/705C12Q2561/113C12Q2563/107C12Q2545/114C12Q2549/119C12Q2531/113C12Q2545/113
Inventor 刘贵明疏义林王绪敏
Owner BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION
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