Nucleic acid for rapidly performing typing detection on avian metapneumovirus subgroup with RT-PCR and application of nucleic acid

An avian metapneumovirus and RT-PCR technology, which is applied to RT-PCR rapid typing and detection of nucleic acid and application fields of avian metapneumovirus subtypes, can solve the problem that indirect enzyme-linked immunosorbent reaction cannot be used as a diagnostic basis, and fluorescent antibodies are not suitable for Large-scale application, unsuitable for large-scale application, etc., to avoid false positive and false negative results, save operation time, and improve detection efficiency

Active Publication Date: 2018-04-13
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The virus isolation method is time-consuming and complicated to operate; the two-step RT-PCR method takes a long time, and the nucleic acid probe technology method and loop-mediated isothermal amplification technology are more expensive
Virus neutralization experiments are time-consuming and expensive, and are not suitable for large-scale applications, and fluorescent antibodies are not suitable for large-scale applications; indirect enzyme-linked immunosorbent assays cannot be used as a basis for diagnosis, and are mainly used for monitoring antibodies in chicken flocks
The above method cannot realize whether to be infected by which subtype among the four subtypes A, B, C, and D of avian metapneumovirus
And the A, B, C, D four subtypes of avian metapneumovirus can realize the method of detecting and typing at the same time.

Method used

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  • Nucleic acid for rapidly performing typing detection on avian metapneumovirus subgroup with RT-PCR and application of nucleic acid
  • Nucleic acid for rapidly performing typing detection on avian metapneumovirus subgroup with RT-PCR and application of nucleic acid
  • Nucleic acid for rapidly performing typing detection on avian metapneumovirus subgroup with RT-PCR and application of nucleic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] The design of embodiment 1 primer

[0043] The present invention adopts reverse transcription polymerase chain reaction (RT-PCR) to detect, and in RT-PCR, an RNA strand is reverse transcribed into complementary DNA, and then DNA amplification is performed by PCR using this as a template. This technology is more sensitive and faster than conventional virus isolation in detecting aMPV, and it is more affordable than fluorescent quantitative RT-PCR.

[0044] In the present invention, the G gene and M gene of avian metapneumovirus are selected as the target genes for detection, because the G gene and M gene are highly conserved within subtypes and have strong specificity between subtypes.

[0045] Using reverse transcription polymerase chain reaction (RT-PCR), avian metapneumovirus is typed, detected and identified, and can detect four subtypes simultaneously in the same sample, and the design of its primer pair is the success of the present invention The key is that there...

Embodiment 2

[0050] Example 2 RT-PCR rapid typing detection

[0051] The method for detecting four subtypes of avian metapneumovirus rapidly and simultaneously genotyped by RT-PCR specifically comprises the following steps:

[0052] (1) Prepare the RNA of the test sample;

[0053] (2) Perform one-step RT-PCR amplification on the RNA sample in step (1): use 20 μL for the reaction system. In a 20 μL reaction system, including: primer pair 1 for avian metapneumovirus type A, primer pair 2 for avian metapneumovirus type B, primer pair 3 for avian metapneumovirus type C, and primer pair for avian metapneumovirus type D The concentration of each primer in 4 can be 10pmol, the dosage is 0.5μL, 5×Buffer 4μL, 2.5mM dNTPs 1.5μL, 5U / μL mixed enzyme (reverse transcriptase and DNA polymerase) 1μL, sample RNA 2μL, dd h 2 O 10.5 μL. All of the above were added into a 0.2mL reaction tube and fully uniform. Then put it into the PCR machine and perform the following reactions: reverse transcription at ...

Embodiment 3

[0064] The detection of embodiment 3 sensitivity

[0065] The RNA quality control product transcribed in vitro of aMPV / C subtype [only the sequence of SEQ ID No.11] is serially diluted by 10 times, detected by the method in Example 2, and analyzed by 1.0% agarose gel electrophoresis , to detect its sensitivity. Its electropherogram is as figure 1 As shown, where M: 2000bp DNAMarker, spotting 1-10 sequence 3.6×10 3 , 3.6×10 2 , 3.6×10, 3.6×10 0 , 3.6×10 -1 , 3.6×10 -2 , 3.6×10 -3 , 3.6×10 -4 , 3.6×10 -5 , 3.6×10 -6 ng / μL, water. The results show that the initial concentration of aMPV / C in vitro transcribed RNA is 3639.4ng / μL. After 10-fold dilution, it is diluted to 1000 times, and the concentration is 3.6ng / μL, which can be detected well, which proves that the sensitivity of the detection method is very good ( See figure 1 shown).

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Abstract

The invention belongs to the technical field of biology and particularly discloses a nucleic acid for rapidly performing typing detection on avian metapneumovirus subgroup with RT-PCR. The nucleic acid is characterized by comprising a primer pair 1 used for detecting avian metapneumovirus A, a primer pair 2 used for detecting avian metapneumovirus B, a primer pair 3 used for detecting avian metapneumovirus C and a primer pair 4 used for detecting avian metapneumovirus D, wherein sequences of the primer pair 1 are shown as SEQ ID No. 1 and SEQ ID No. 2, sequences of the primer pair 2 are shownas SEQ ID No. 3 and SEQ ID No. 4, sequences of the primer pair 3 are shown as SEQ ID No. 5 and SEQ ID No. 6, and sequences of the primer pair 4 are shown as SEQ ID No. 7 and SEQ ID No. 8. The nucleicacid can be used for respectively detecting an avian metapneumovirus subgroup A, an avian metapneumovirus subgroup B, an avian metapneumovirus subgroup C and an avian metapneumovirus subgroup D, the used primers are strong in specificity, no cross reaction exists among the primer pairs, and the primer pairs does not have cross reaction with other viruses.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a nucleic acid used for RT-PCR rapid typing to detect subtypes of avian metapneumovirus and its application. Background technique [0002] Avian metapneumovirus (aMPV), also known as avian metapneumovirus or turkey rhinotracheitis virus, is an important pathogen of poultry distributed worldwide. The virus can cause acute upper respiratory tract infection in poultry, head swelling of adult chickens, lower egg production rate and hatchability of infected laying hens. Avian metapneumovirus belongs to the family Paramyxoviridae and the genus Metapneumovirus in the subfamily Pneumoviridae. It is a non-segmented single-stranded negative-sense RNA virus with a length of about 14 kb. It consists of 8 genes, mainly encoding 8 structural proteins, in order For: 3' leader (Leader), nucleoprotein (N), phosphoprotein (P), matrix protein (M), fusion protein (F), second matrix protein ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/701C12Q2600/166C12Q2545/113C12Q2521/107
Inventor 刘爵王利佳姜海军韦莉王菁全荣朱珊珊侯磊王丹李子璇
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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