Infectious hematopoietic necrosis virus detection kit based on pyrosequencing

A technology for hematopoietic organ necrosis and pyrosequencing, which can be applied in microorganism-based methods, microorganism determination/inspection, microorganisms, etc., and can solve problems such as cumbersome operations.

Inactive Publication Date: 2015-12-02
INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the traditional virus isolation and serological diagnosis methods are cumbersome and cannot realize the simultaneous detection of different subtypes of viruses. An integrated diagnostic technology capable of high-throughput rapid screening of SVCV, VHSV and IHNV

Method used

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  • Infectious hematopoietic necrosis virus detection kit based on pyrosequencing
  • Infectious hematopoietic necrosis virus detection kit based on pyrosequencing
  • Infectious hematopoietic necrosis virus detection kit based on pyrosequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1. Preparation of primers for detection of carp spring viremia virus, viral hemorrhagic sepsis virus and infectious hematopoietic organ necrosis virus

[0073]The material for detecting the fingerprint sequence of carp spring viremia virus (SVCV) includes a sequencing primer SVCV-S for the fingerprint sequence of carp spring viremia virus and a pair of primers SVCV-P for amplifying the fingerprint sequence of carp spring viremia virus. The fingerprint sequence of the carp spring viremia virus is nucleotides 511-526 of SEQ ID No. 1 in the sequence listing. The SVCV-S is the single-stranded DNA shown in SEQIDNo.4 in the sequence listing; the SVCV-P is the single-stranded DNA (SVCV-P-F) shown in SEQIDNo.2 and SEQIDNo.3 in the sequence listing A pair of primers composed of single-stranded DNA (SVCV-P-R), the 5' end of the SVCV-P-R is labeled with biotin, and the amplification product of SVCV-P is the 454th-591st nucleotide of SEQIDNo.1 in the sequence table For the...

Embodiment 2

[0078] The optimization of embodiment 2, SVCV-P, VHSV-P and IHNV-P amplification conditions

[0079] 1. Construction of recombinant vector

[0080] Replace the sequence between the BamHI and HindIII recognition sites of the pMD18-Tsimple vector with the nucleotide sequence shown in SEQIDNo. The vector was named PMD18-T-SVCV.

[0081] Replace the sequence between the HindIII and XhoI recognition sites of the pMD18-Tsimple vector with the nucleotide sequence shown in SEQIDNo. The vector was named PMD18-T-VHSV.

[0082] Replace the sequence between the HindIII and BamHI recognition sites of the pMD18-Tsimple vector with the nucleotide sequence shown in SEQIDNo. The vector was named PMD18-T-IHNV.

[0083] 2. Optimization of SVCV-P amplification conditions

[0084] The amplification conditions of SVCV-P were optimized with the following PCR amplification system: 2 μL of PMD18-T-SVCV vector at a concentration of 50 ng / μl, 10 μL of 5×PCR buffer, 0.25 μL of 5 U / μl Taq hot-start e...

Embodiment 3

[0092] Embodiment 3, identification of SVCV, VHSV and IHNV by pyrosequencing

[0093] The RNAs of SVCV, VHSV and IHNV were extracted to obtain SVCV total RNA, VHSV total RNA and IHNV total RNA at a concentration of 40 ng / μL, respectively.

[0094] 1. Identification of SVCV by pyrosequencing

[0095] 1.1 PCR amplification

[0096] 50 μL PCR amplification system: 2 μL of SVCV total RNA at a concentration of 40 ng / μL, 10 μL of 5×PCR buffer, 0.25 μL of 5 U / μL Taq hot start enzyme, 0.5 μL of SVCV-P-F at a concentration of 10 pmol / μL, and SVCV-P-F at a concentration of 10 pmol / μL P-R 0.5 μL, make up the volume to 50 μL with DEPC water.

[0097] The amplification conditions were: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, and 50 cycles; extension at 72°C for 10 min, and ending at 4°C.

[0098] The obtained PCR products were subjected to agarose gel electrophoresis ( figure 1 ), the results showed that...

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Abstract

The invention discloses an infectious hematopoietic necrosis virus detection kit based on pyrosequencing. The infectious hematopoietic necrosis virus detection kit based on the pyrosequencing depends on an infectious hematopoietic necrosis virus fingerprint sequence, and the infectious hematopoietic necrosis virus fingerprint sequence is nucleotides at the 553th-565th positions of SEQ ID No.9 shown in a sequence table. The kit comprises a sequencing primer of the infectious hematopoietic necrosis virus fingerprint sequence and a primer pair for amplifying the infectious hematopoietic necrosis virus fingerprint sequence. The sequencing primer is single-stranded DNA represented by SEQ ID No.12 shown in the sequence table named as IHNV-S. The primer pair for amplifying the infectious hematopoietic necrosis virus fingerprint sequence consists of single-stranded DNA represented by SEQ ID No.10 shown in the sequence table named as IHNV-P and single-stranded DNA represented by SEQ ID No.11 shown in the sequence table.

Description

technical field [0001] The invention relates to a detection kit for infectious hematopoietic organ necrosis virus based on pyrosequencing in the field of biotechnology. Background technique [0002] Carp spring viremia (SVC), viral hemorrhagic sepsis (VHS), and infectious hematopoietic necrosis (IHN) are listed in the "List of Class I and Class II Infectious and Parasitic Diseases of Imported Animals of the People's Republic of China" 》The second-class infectious diseases, SVC, VHS and IHN are the second-class and third-class epidemic diseases stipulated in the "Animal Epidemic Prevention Law of the People's Republic of China" respectively. These three diseases are all important diseases in the OIE list of the International Office of Epizootics. [0003] VHS has plagued European rainbow trout for more than 50 years, and is considered to be the number one killer of economic losses in the European rainbow trout farming industry. VHS has gradually been reported in other species...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/701C12Q1/6869C12Q2531/113C12Q2535/122C12Q2565/301
Inventor 尹伟力姜华张晓文鲁闽刘宁岳志芹
Owner INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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