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RT-PCR detection primers and detection method of subgroup k avian leukosis virus

An avian leukemia virus, RT-PCR technology is applied to the K subgroup avian leukemia virus RT-PCR detection primer and detection field, which can solve the problems of easy contamination, high cost of detection reagents and consumables, inconvenience for promotion and application on breeding sites, and achieves specificity. The effect of high performance, low detection cost, and easy configuration

Active Publication Date: 2021-10-26
WENS FOODSTUFF GRP CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Patent 1 (Application No.: CN201710093589.8) and Patent 2 (Application No.: CN201810074475.3) disclose a fluorescent quantitative PCR detection method for K subgroup avian leukosis virus. The detection sensitivity of this method is high, but this method requires the use of fluorescent quantitative PCR Instruments and other precision instruments and equipment, the cost of detection reagents and consumables is relatively high, and it is easy to pollute in the ordinary experimental environment, which is not convenient for promotion and application in the breeding field

Method used

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  • RT-PCR detection primers and detection method of subgroup k avian leukosis virus
  • RT-PCR detection primers and detection method of subgroup k avian leukosis virus
  • RT-PCR detection primers and detection method of subgroup k avian leukosis virus

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Experimental program
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Effect test

Embodiment 1

[0026] Embodiment 1 K subgroup avian leukosis virus RT-PCR detection primer and detection method of the present invention

[0027] 1 test material

[0028] 1.1 Main reagents and instruments

[0029] AxyPrep Body Fluid Viral DNA / RNA Mini Kit (Cat No.: AP-MN-BF-VNA-250), TaKaRaPrimeScript 0ne step RT-PCR Kit Ver.2 (TaKaRa Code: DRR055A), Thermo High Speed ​​Low Temperature Centrifuge, Thermo PCR instrument, electrophoresis instrument.

[0030] 1.2 Strains

[0031] Clinically identified avian leukosis virus by virus isolation and ELISA detection, clinically identified avian leukosis virus of A, B, J and K subgroups.

[0032] 1.3 Primer design

[0033] Design a pair of primers according to the env gene sequence of K subgroup virus GD14LZ strain (GenBank: KU605774.1) as shown in the table below to amplify part of the gene fragment. The expected size of the target band is 401bp. If the target fragment is amplified, it can be determined as subgroup K avian leukemia virus.

[00...

Embodiment 2

[0046] Example 2 specificity verification

[0047] Samples were used: negative control; samples positive for p27 detected by ELISA in production; subgroup K virus as positive control; subgroup A, B, and J viruses.

[0048] Test method: According to the detection method in Example 1, each sample was amplified respectively, and identified and compared by agarose gel electrophoresis respectively.

[0049] Test results: In the samples that were detected as p27 positive by ELISA in production, the K subgroup virus could amplify the target fragment, and the A, B, J subgroup viruses and other subgroup viruses isolated in production could not amplify the target fragment ( like figure 2 ).

Embodiment 3

[0050] Example 3 Sensitivity Verification

[0051] Samples used: negative control; K subgroup avian leukosis virus.

[0052] Test method: extract K subgroup avian leukosis virus sample RNA according to the detection method of embodiment 1, measure RNA concentration, and RNA is carried out 10 times ratio dilutions, each dilution RNA is amplified respectively, and respectively through agarose coagulation Gel electrophoresis identification comparison.

[0053] For test results, see image 3 : The sample RNA concentration is 95.07ng / μl, and the sample is undiluted and 10 -1 、10 -2 、10 -3 The target fragment can be amplified at all dilutions, and the concentration of the amplified product decreases sequentially. samples in 10 -4 The dilution cannot amplify the target fragment.

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Abstract

The invention relates to the technical field of molecular biology detection, in particular to a RT-PCR detection primer and a detection method for K subgroup avian leukosis virus. The primers of the present invention are represented by SEQ ID NO:1 and SEQ ID NO:2. The RT-PCR detection method of K subgroup avian leukosis virus of the present invention comprises the following operations: (1) RNA template preparation; (2) amplifying the target fragment; (3) identifying the amplified product by electrophoresis. The primers of the present invention have high specificity and short extension fragments, and the entire PCR identification process can be completed within 2 to 2.5 hours, and the K subgroup avian leukosis virus can be quickly identified without sequencing. The diagnosis saves a lot of time. The method of the invention is simple to operate, the equipment used in ordinary experiments is easy to configure, the detection cost is low, and it is convenient for clinical popularization and application.

Description

technical field [0001] The invention relates to the technical field of molecular biology detection, in particular to an RT-PCR detection primer and a detection method for K subgroup avian leukosis virus. Background technique [0002] A, B and J subgroups of avian leukemia are mainly prevalent in chicken flocks in my country, and K subgroup avian leukemia is a new subgroup isolated from Chinese local breeds in recent years. At present, the identification of avian leukosis virus is mainly through plasma virus isolation and ELISA technology in production, but this method cannot determine the virus subgroup. A, B, and J subgroup viruses can be identified by PCR with specific primers, while K subgroup viruses must be amplified with general primers for avian leukosis virus, sequenced and sequenced to be identified, which takes a long time and gives K subgroup viruses a fast Identification is inconvenient. [0003] At present, there are two patents related to the detection of sub...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/702C12Q2521/107
Inventor 严专强刘洋明东怡王占新鲁俊鹏覃健萍廖秋生
Owner WENS FOODSTUFF GRP CO LTD
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