Real-time quantitative PCR detection method for red-sea bream iridovirus

A real-time quantitative technology of red sea bream iridescent virus, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, fluorescence/phosphorescence, etc. The effect of sample cross-contamination, reducing environmental pollution and wide detection range

Inactive Publication Date: 2010-09-29
INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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Problems solved by technology

The third is immunological methods: According to the OIE Aquatic Animal Diseases Diagnostic Manual, immunological methods can diagnose different genera of iridescent viruses, but there are often cross-reactions between genera; because most iridescent viruses cannot obtain sufficient anti-specific iridescent viruses by immunizing rabbits, etc. Neutralizing antibodies to the virus, so neutralization tests cannot be used to identify iridescent viruses; indirect fluorescent antibody test (IFAT) and enzyme-linked immunoassay (ELISA) are adopted as OIE-approved diagnostic methods and their specific operations are specified ;Antigen capture ELISA can replace virus isolation as a way to diagnose infectious hematopoietic necrosis virus (EHNV), but its sensitivity and specificity vary for different hosts
The fourth is molecular biology methods: OIE also cautiously accepts PCR as a diagnostic method, using a conserved fragment of the DNA polymerase gene encoding red sea bream iridescent virus to design primers, which can be obtained from cell lines of aquatic animals infected with iridescent viruses and related Target fragments are amplified in tissues (liver, spleen, brain, kidney), and the amplified fragments need to be sequenced to further confirm the amplification results, but at the same time, positive and negative controls must be set up to prevent false positives and false positives. Negative, this method can only be used for qualitative detection, not quantitative detection
To sum up, the existing detection methods generally have the disadvantages of complex detection steps, poor sensitivity, difficult control of the operation process, low accuracy of detection results, and difficult quantitative analysis.

Method used

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  • Real-time quantitative PCR detection method for red-sea bream iridovirus
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  • Real-time quantitative PCR detection method for red-sea bream iridovirus

Examples

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Embodiment 1

[0017] Taking perch samples as an example, real-time quantitative PCR method with fluorescent probes was used to quantitatively detect red sea bream iridescent virus; sea bass samples were collected from a farm in Shandong Province, and had typical clinical symptoms of red sea bream iridescent virus infection, which appeared on the skin, fins and eyeballs of the fish. Small vesicle-like swellings, with medical history of ciliates, tympanum, etc.; the above-mentioned designed specific primer sequences and fluorescent probe sequences:

[0018] In http: / / www.ncbi.nlm.nih.gov / Genbank / , search for the sequence of red snapper iridescent virus, and use the existing DNALASERGENE 7.1 software to analyze and compare the conservation of each gene sequence. The results show that the main red snapper iridescent virus is The capsid protein (MCP) gene has better conservation and research value, and is a major trend in virus research at present. Therefore, using the MCP gene as the target gen...

Embodiment 2

[0035] Taking the perch sample as an example, utilize SYBR GREEN I fluorescent dye to detect whether the perch sample contains red sea bream iridescent virus; Under the circumstances, there is no need to design a fluorescent probe; the real-time quantitative PCR reaction system and reaction procedure are the same as in Example 1, except that 1 μL of 10 μmol / L fluorescent probe in the real-time quantitative PCR reaction system is replaced with 1 μL of 25×SYBR GREEN I (ROCHE) fluorescent dye. Moreover, after the real-time quantitative PCR reaction program is completed, use the ABI 7900HT quantitative PCR instrument for melting curve analysis. The program is 95°C for 15 minutes, 60°C for 15 minutes, and 95°C for 15 minutes. The heating and cooling rate is 1°C per 25 seconds. Finally, the Tm value of the sample is given by the SDS 2.1 software; the instrument collects the fluorescence signal, and the analysis data is the same as in Example 1; the melting curve of the detection sam...

Embodiment 3

[0037] To analyze the specificity of SYBR GREEN I real-time quantitative PCR detection method for red sea bream iridescent virus; as a control, other viruses that are closely related to red sea bream iridescent virus were used for detection by SYBR GREEN I real-time quantitative PCR method, and these viruses belonged to Iridoviridae, they are epidemic hematopoietic necrosis virus (EHNV), lymphocyst virus (LCDV), frog virus 3 (FV 3), soft shell turtle iridescent virus (STIV); detection step is the same as embodiment 2, just will treat The DNA of the test sample was replaced with the DNA of epidemic hematopoietic necrosis virus (EHNV), lymphocyst virus (LCDV), frog iridescent virus (BIV), soft shell turtle iridescent virus (STIV); melting curve of SYBR GREEN I real-time quantitative PCR See Figure 5 Among them, the Tm value of the amplified product of red sea bream iridescent virus was 82.2°C, the Tm value of the other four viruses exceeded the range of 82.0°C-830, and the fluo...

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Abstract

The invention belongs to the technical field of detection of red-sea bream iridovirus infection, and relates to a real-time quantitative detection method of polymerase chain reaction (PCR) technology by adopting a fluorescent probe or an SYBR GREEN I fluorescent dye, in particular to a real-time quantitative PCR detection method for red-sea bream iridovirus. The method comprises the following steps of: designing a specific primer sequence and a fluorescent probe sequence, then establishing a real-time quantitative PCR system and a reaction program, preparing plasmids containing a target amplification fragment as a standard substance to draw a standard curve, and finally establishing a result judgment base of a sample to be detected. The method has the advantages of simple detection, high detection speed, good effect, accurate quantification, strong specificity, high sensitivity and the like.

Description

Technical field: [0001] The invention belongs to the technical field of detecting red sea bream iridescent virus infection, and relates to a detection method using a fluorescent probe or a SYBRGREEN I fluorescent dye for real-time quantitative polymerase chain reaction (PCR) technology, in particular to a red sea bream iridescent virus infection real-time quantitative PCR detection method. Background technique: [0002] Red-sea bream iridovirus (Red-sea bream iridovirus, RSIV) belongs to the Iridoviridae family and belongs to the genus Cytometavirus. Affect the import and export trade of economic fish. In view of the harmfulness of the iridescent virus, the RSIV iridescent virus is listed as a list of OIE quarantine diseases. In 2008, my country newly announced the "List of Types I, II, and III Animal Diseases" (Announcement No. 1125 of the Ministry of Agriculture of the People's Republic of China) It is classified as a second-class disease. South Korea has implemented the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/64
Inventor 赵玉然岳志芹赵巍郑小龙邓明俊朱来华徐彪梁成珠
Owner INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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