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Primer composition for detecting mycoplasma sheep pneumoniae and peste des petits ruminants virus, kit and application

A technology for Mycoplasma pneumoniae and Peste des petits ruminants, which is applied in the fields of biotechnology and molecular biology, can solve the problems of long detection time, unfavorable clinical rapid diagnosis, long time consumption, etc., and achieves the effects of saving time, convenient and rapid detection, and simple operation.

Active Publication Date: 2019-05-31
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Mycoplasma isolation culture and virus isolation are commonly used methods to detect pathogens, but they take a long time and are not conducive to rapid clinical diagnosis. Compared with them, PCR and RT-PCR methods are simpler and faster, and are suitable for laboratory diagnosis
However, currently there are only PCR and RT-PCR methods for the detection of MO and PPRV alone, and the detection takes a long time, and there is no report and application of a method for simultaneously detecting MO and PPRV

Method used

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  • Primer composition for detecting mycoplasma sheep pneumoniae and peste des petits ruminants virus, kit and application
  • Primer composition for detecting mycoplasma sheep pneumoniae and peste des petits ruminants virus, kit and application
  • Primer composition for detecting mycoplasma sheep pneumoniae and peste des petits ruminants virus, kit and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Primer Design and Double RT-PCR Inspection

[0024] 1. Primer design and synthesis

[0025] Primer design is the key to the success of multiplex PCR. In the present invention, by analyzing and comparing MO and PPRV gene sequences, a variety of target genes are selected from the two viral genes to design primers, and viral DNA and RNA are used as templates to amplify by RT-PCR to ensure that the correct In the case of identifying two pathogens, it is also necessary to avoid missed detection of pathogens of the same species and different strains.

[0026] The applicant used the MPprimer software, a multiplex PCR primer design system, to design nearly a hundred pairs of primer combinations for MO and PPRV genes, in order to screen the best primer combinations and detect the two pathogens at the same time. When designing multiple primers, in addition to considering factors such as the Tm value, GC content, and primer specificity of a single pair of primers, it i...

Embodiment 2

[0054] Example 2 Establishment of double RT-PCR detection method

[0055] 1. Preparation of Standard Plasmids

[0056] Take 200 μL MO culture solution (10 7 CCU / mL, Y98 strain, purchased from China Veterinary Drug Administration) was placed in a 1.5mL EP tube (RNase free), nucleic acid was extracted, and PCR amplification was performed using primer pair A. The reaction system was: in a 0.2 ml PCR reaction tube Add 10 μL of 2×PCR Mix, 0.5 μL of each primer, 2 μL of nucleic acid template, add sterile double distilled water to a total volume of 20 μL, and mix well. The reaction program was: 94 °C pre-denaturation for 5 min; 94 °C for 30 s, 54 °C for 30 s, 72 °C for 30 s, a total of 35 cycles; finally, 72 °C extension for 10 min. According to the electrophoresis observation results, the PCR product with a size of 359 bp was recovered and purified, and cloned into the pMD18T vector. The correct positive plasmid after sequencing was the MO standard plasmid.

[0057] Take 200 μL ...

Embodiment 3

[0063] Example 3 kit assembly and sample detection

[0064] 1. Kit Assembly

[0065] The double RT-PCR detection kit (abbreviated as the kit of the present invention) for detecting Mycoplasma ovis pneumoniae and Peste des Petits Ruminants virus includes the following reagents:

[0066] (1) Primers

[0067] The primers include primer 1, primer 2, primer 3 and primer 4 (same as Example 1), and the concentration of each primer is 10 μmol / L.

[0068] (2) 2×R-Mix Buffer

[0069] 2×R-Mix Buffer was purchased from Beijing Quanshijin Biotechnology Co., Ltd., and it is a product in the EasyScript One-Step RT-PCR SuperMix kit.

[0070] (3) E-Mix

[0071] E-Mix was purchased from Beijing Quanshijin Biotechnology Co., Ltd., and is a product in the EasyScript One-Step RT-PCR SuperMix kit.

[0072] (4) RNase-free double distilled water

[0073] (5) Positive standard

[0074] Positive standard contains 1 × 10 6 copies / μL of MO standard plasmid and 1 x 10 6 copies / μL of PPRV

[00...

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Abstract

The invention provides a primer composition for detecting mycoplasma sheep pneumoniae and peste des petits ruminants virus, a kit and application, and belongs to the field of biotechnology and molecular biology. The primer composition for detecting mycoplasma sheep pneumoniae and peste des petits ruminants virus includes a primer pair A and a primer pair B, the primer pair A includes a primer 1 and a primer 2, the primer pair B includes a primer 3 and a primer 4, and the nucleotide sequences of the primers 1-4 are shown as SEQIDNO:1-4. The primer composition can be used for detecting mycoplasma sheep pneumoniae and peste des petits ruminants virus simultaneously or individually, operation is simple, the specificity is high, the sensitivity is high, and the detection efficiency is remarkably improved. The situation is avoided that one pathogen can only be detected at a time, the time is saved, pollution is avoided, rapid detection of clinical respiratory disease samples is facilitated,and the primer composition has broad application prospects.

Description

technical field [0001] The invention belongs to the fields of biotechnology and molecular biology, and in particular relates to a primer composition, a kit and an application for detecting mycoplasma ovis pneumonia and Peste des Petits Ruminants virus. Background technique [0002] Mycoplasma ovipneumoniae (MO) is one of the main pathogens causing respiratory diseases in sheep and can cause atypical pneumonia in goats, sheep and some wild ruminants. Not only that, the susceptibility of the host to other pathogens is enhanced after infection with M. ovis. The pathogen is currently distributed all over the world, causing large economic losses to the sheep industry. Peste des petits ruminants virus (PPRV) is a member of the Morbillivirus genus of the family Paramyxovirinae, which can cause fever, large amounts of secretions from the eyes and nose, pneumonia, Goats with clinical symptoms such as upper gastrointestinal ulcers and diarrhea, and subclinical infections can continu...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/689C12Q1/686C12Q1/04C12N15/11C12R1/35C12R1/93
Inventor 李文良霍晓丽毛立杨蕾蕾刘茂军郝飞李基棕孙敏
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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