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RPA (recombinase polymerase amplification) primer pair, probe, kit and detection method for rapidly detecting Marek's disease virus (MDV)

A Marek's disease and primer pair technology, applied in the field of molecular biology detection, achieves the effects of high sensitivity, strong amplification specificity, and wide application prospects

Active Publication Date: 2020-01-17
广州千寻生物技术有限公司 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the detection of MDV by real-time fluorescent RPA at home and abroad.

Method used

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  • RPA (recombinase polymerase amplification) primer pair, probe, kit and detection method for rapidly detecting Marek's disease virus (MDV)
  • RPA (recombinase polymerase amplification) primer pair, probe, kit and detection method for rapidly detecting Marek's disease virus (MDV)
  • RPA (recombinase polymerase amplification) primer pair, probe, kit and detection method for rapidly detecting Marek's disease virus (MDV)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1. Establishment of a Recombinase Polymerase Amplification RPA Method for Rapid Detection of Marek's Disease Virus

[0033] 1. Materials and methods

[0034] 1. Virus Extraction

[0035] According to the instructions of the TIANamp Genomic DNAKit Blood / Cell / Tissue Genomic DNA Extraction Kit from TIANGEN Company, the DNA was extracted from the feather marrow samples of sick chickens. Add 200 µL of virus solution to a 1.5 mL centrifuge tube, add 20 µL of proteinase K and 200 µL of carrier RNA solution, shake the mixture and mix. After 15 min in a 56 °C water bath, add 250 μL of absolute ethanol, transfer to a tube, and centrifuge at 8000 rpm for 1 min. The wash solution was washed twice, then TE was added, left at room temperature for 5 min and centrifuged to obtain DNA / RNA.

[0036] 2. Cloning of target gene

[0037] Meq gene cloning was performed using conventional methods. The meq gene was amplified by conventional PCR. The amplification system was: 95°C / 1...

Embodiment 2

[0054] Embodiment 2. Specificity Test and Sensitivity Test of Recombinase Polymerase Amplification RPA Method for Rapid Detection of Marek's Disease Virus in the Present Invention

[0055] Specificity test: by testing for MDV with positive nucleic acid as well as other nucleic acids from other pathogens, including Newcastle disease virus (NDV), avian infectious anemia virus (CAV), infectious bursal disease virus (IBDV), avian infectivity Bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), avian influenza virus (AIV) and avian leukosis virus (ALV) and avian reovirus (ARV) were used to determine the specificity of the constructed real-time RPA method. wxya 2 O as a blank control.

[0056] Specific test results such as figure 2 It was shown that, except for the amplification of MDV, other negative nucleic acids were not amplified, proving that the method of the present invention has good specificity and no cross-reaction with other pathogens.

[0057] Sensitivi...

Embodiment 3

[0059] Embodiment 3, the present invention rapid detection of Marek's disease virus recombinase polymerase amplification RPA method for the detection of clinical samples

[0060] In order to evaluate the practicability of the MDV real-time RPA method constructed in the present invention, 25 clinical samples suspected of MDV infection were detected and compared using the real-time PCR assay. The results showed (Table 2) that the positive detection rate of RPA in 25 clinical samples suspected of MDV infection was 32% (8 / 25), which was consistent with the positive detection rate of real-time PCR. The concordance of positive and negative samples for both methods was 100%.

[0061] Table 2

[0062] Detection method positive sample negative sample The detection rate RPA 8 17 32％ PCR 8 17 32％

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Abstract

The invention provides an RPA (recombinase polymerase amplification) primer pair, probe, kit and detection method for rapidly detecting Marek's disease virus (MDV), and belongs to the technical fieldof molecular biological detection. Sequences of the RPA primer pair are shown in SEQ ID No.1 and SEQ ID No.2; and a sequence of the RPA probe is shown in SEQ ID No.3. The detection method can completedetection at 39 DEG C within 20 min, has the characteristics of being rapid, sensitive, convenient to operate and applicable to laboratories and field rapid detection, and provides a technical reference for rapid diagnosis, prevention and control of the MDV.

Description

technical field [0001] The invention relates to an RPA primer pair, a probe, a kit and a detection method for rapidly detecting Marek's disease virus, and belongs to the technical field of molecular biology detection. Background technique [0002] Marek's Disease Virus (Marek`s Disease Virus, MDV), also known as neurolymphomatosis, belongs to the family Herpesviridae, Alphaherpesvirinae, Marekvirus genus, can cause Marek`s Disease (Marek`s Disease), lymphoma A proliferative disease that mainly affects poultry such as chickens, turkeys, pheasants, and quails. Clinically, it is characterized by single or multiple infiltration of lymphoid cells in visceral organs, peripheral nerves, gonads, iris, muscles and skin, and the apparent symptoms of infected Marek's disease chickens may be paralysis or severe or even death . The virus of this disease exists in the epithelial cells of the feather follicle in large quantities, so the virus detection can be carried out by detecting the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/93
CPCC12Q1/705C12Q1/6844C12Q2521/507C12Q2563/107C12Q2522/101C12Q2561/113Y02A50/30
Inventor 丛锋刘长军张艳萍张桐源
Owner 广州千寻生物技术有限公司
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