Fast detection primer, reagent kit and method for channel catfish viruses and application of fast detection primer, reagent kit and method

A technology of catfish herpes virus and channel catfish, applied in biochemical equipment and methods, microbiological measurement/testing, DNA/RNA fragments, etc., can solve the problems of great harm, long detection cycle, cumbersome operation, etc., and achieve good specificity , fast detection and simple operation

Inactive Publication Date: 2019-12-31
HOHAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

With the continuous expansion of the scale of channel catfish farming, the increasing degree of intensification, and the deterioration of the breeding environment, channel catfish diseases are becoming more and more serious, especially Channel Catfish Virus Disease (CCVD) caused by channel catfish virus (CCV) The most harmful, causing serious economic losses to the channel catfish farming industry
At present, the traditional detection methods of CCVD mainly use cell culture technology to diagnose CCVD, and then combine serum neutralization test, fluorescent antibody technology, ELISA or PCR identification. Shortcomings such as a long detection cycle cannot meet the actual work needs of rapid diagnosis of pathogens and timely control of the epidemic

Method used

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  • Fast detection primer, reagent kit and method for channel catfish viruses and application of fast detection primer, reagent kit and method
  • Fast detection primer, reagent kit and method for channel catfish viruses and application of fast detection primer, reagent kit and method
  • Fast detection primer, reagent kit and method for channel catfish viruses and application of fast detection primer, reagent kit and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 : Establishment of a channel catfish CCVD pathogen detection method using a real-time fluorescence quantitative instrument:

[0029] The rapid detection method for channel catfish herpes virus disease pathogen includes PCR primer set design, DNA template preparation, PCR amplification and result judgment by fluorescent signal.

[0030] (1) PCR primer and probe design: According to the channel herpes virus type I gene sequence published in NCBI, Primer 5.0 software was used to design primers and probes for the conserved sequence of the ORF73 gene of CCV virus as the target. The designed primers and The probe sequences are shown in Table 1.

[0031] Table 1. PCR primer and probe sequences

[0032]

[0033] (2) positive control is the plasmid DNA containing channel catfish CCVD pathogen toga protein gene ORF73 fragment, and its preparation method is: extract channel catherpes virus DNA, utilize the outer primers (SEQ ID NO: 1 and SEQ ID NO: 1 and SEQ ID NO:...

Embodiment 2

[0038] Example 2 : Detection specificity experiment:

[0039] The method of Example 1 was used to detect the positive DNA of channel herpes virus and the DNA of koi herpes virus, frog iridovirus and mandarin fish infectious spleen and kidney necrosis virus respectively.

[0040] Identification results such as figure 1 Shown: The rapid detection method of channel catfish CCVD pathogen was used for the amplification reaction, the positive DNA of channel catfish virus was amplified normally, and the negative water control and koi herpes virus, frog iris virus, mandarin fish infectious spleen and kidney necrosis virus did not amplify, Shows good specificity.

Embodiment 3

[0041] Example 3 : Detection Sensitivity Experiment:

[0042] Quantify the plasmid containing the ORF73 fragment of the oncoprotein gene of the CCVD pathogen, determine its concentration and calculate the plasmid copy number according to the molecular weight, and dilute to 1.6×10 7 copies / μl, 1.6×10 6 copies / μl, 1.6×10 5 copies / μl, 1.6×10 4 copies / μl, 1.6×10 3 copies / μl, 1.6×10 2 copies / ul,1.6×10 1 copies / μl, 1.6×10 0 copies / μl. The diluted positive clones were detected respectively by the operation method of Example 1. Identification results such as figure 2 Shown: The limit of detection for positive plasmids reached 1.6 × 10 1 copies / μl;

[0043] Quantify the plasmid containing the ORF73 fragment of the oncoprotein gene of the CCVD pathogen, determine its concentration and calculate the plasmid copy number according to the molecular weight, and dilute to 1.6×10 7 copies / μl, 1.6×10 6 copies / μl, 1.6×10 5 copies / μl, 1.6×10 4 copies / μl, 1.6×10 3 copies / μl, 1.6×...

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Abstract

The invention discloses a fast detection primer, reagent kit and method for channel catfish viruses and an application of the fast detection primer, the reagent kit and the method, and belongs to thefield of a biological technology. The method comprises steps of designing a PCR primer and a probe, preparing a DNA template, performing PCR amplification and performing result judging through luminescence signals. In accordance with gene conserved sequences corresponding to the channel catfish viruses, the primer and a taqMan probe are designed, and through sufficient optimization, the primer probe and the reaction condition are originally created, so that the method for quickly and reliably detecting the channel catfish viruses through fluorescent quantitation PCR is established. According to the fast detection primer, reagent kit and method, quantification of the DNA template is realized, and the fast detection primer, the reagent kit and the method have the advantages of being simple to operate, quick to detect, good in specificity, high in sensitivity, reliable in results and the like, and can exert important effects in respects of early rapid diagnosis and real-time monitoring ofthe channel catfish viruses.

Description

technical field [0001] The invention belongs to the field of biotechnology, relates to a molecular detection method for pathogens of freshwater aquaculture animals, and in particular relates to a rapid detection primer, a kit, a method for channel catfish herpes virus disease (CCVD) pathogens established by using a taqMan fluorescence quantitative method, and a method thereof. application. Background technique [0002] Channel Catfish Virus (CCV) is an enveloped double-stranded DNA virus, also known as Ictalurid herpesvirus 1, which can cause lethal infection of channel catfish fry and fingerlings. With the continuous expansion of channel catfish breeding scale, the increasing degree of intensification and the deterioration of the breeding environment, the disease of channel catfish is becoming more and more serious, especially the channel catfish virus disease (CCVD) caused by channel catfish virus (CCV). It is the most harmful, causing serious economic losses to the chann...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11
CPCC12Q1/6851C12Q1/705C12Q2600/166C12Q2531/113C12Q2561/101C12Q2545/113
Inventor 郝凯赵哲
Owner HOHAI UNIV
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