Primers, probes and kit for rapidly detecting mycoplasma bovis on site

A technology of Mycoplasma bovis and a kit is applied in the field of microbial detection to achieve the effects of high sensitivity, strong specificity and convenient use

Inactive Publication Date: 2017-06-20
SHANDONG NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] To sum up, for the RPA-LFD detection of Mycoplasma bovis, there are no clear ideas and result

Method used

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  • Primers, probes and kit for rapidly detecting mycoplasma bovis on site
  • Primers, probes and kit for rapidly detecting mycoplasma bovis on site
  • Primers, probes and kit for rapidly detecting mycoplasma bovis on site

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Design and screening of embodiment 1 primers and probes

[0076] At present, there are no specific rules for the design of RPA-nfo primers and probes, and the specificity and amplification efficiency must be tested after the RPA reaction, in order to screen and obtain primers and probes that can be used in clinical testing. In the experiment, it is necessary to design multiple pairs of primers and probes from both ends of the target sequence for optimization and screening, and the substitution or increase or decrease of individual bases will have an important impact on the test results.

[0077] The present invention designs forward primers, reverse primers and probes respectively according to the uvrC gene (GengBank No.AF003959) and oppD-oppF gene (GengBank No.AF130119) specific to Mycoplasma bovis, see Table 1 respectively. When designing primers, the conservation of uvrC gene and oppD-oppF gene in the primer design region was firstly BLASTed on the Genebank data, bot...

Embodiment 2

[0084] The establishment of embodiment 2 Mycoplasma bovis RPA-LFD detection method

[0085] 1. Experimental steps

[0086] (1) Preparation of positive quality control standard:

[0087] ①Extraction of template DNA: Genomic DNA was extracted from the lung tissue of dairy cows positive for Mycoplasma bovis detected by PCR in our laboratory.

[0088] ②PCR amplification: using the extracted DNA as a template, use the upstream primer uvrC-S: 5'-TAAATGAGCGCAGTGCTGAT-3' and the downstream primer uvrC-A: 5'-AACTTGAATTTGAACTAAGT-3' to amplify. The reaction system is 50 μL, including 2 ×PCR mix: 25 μL, 2 μL of upstream and downstream primers, 2 μL of template, ddH 2 O 19 μL. The reaction conditions are: 94°C for 3 min, 94°C for 30 sec, 60°C for 30 sec, 72°C for 30 sec, 35 cycles, 72°C for 10 min.

[0089] ③Construction of recombinant plasmids and establishment of positive quality control standards: PCR products were gel-purified and recovered, then connected to the pEASY-T3 vector (...

Embodiment 3

[0108] Application of embodiment 3 Mycoplasma bovis RPA-LFD detection method

[0109] 1. Experimental steps

[0110] (1) On-site preparation of clinical samples

[0111] Clinical samples such as lung tissue, aerosol centrifugation, and milk sample centrifugation were washed with 200 μL TE buffer [1.0MTris-HCl (pH8.0) 10mL, 0.5M Na 2 EDTA·2H 2 O (pH8.0) 2mL, add distilled water to 1000mL] to resuspend, take 200μL of nasal swab solution, blood and other liquid samples directly, then add 30μL 10% (w / v) SDS and 3μL 2% (w / v) For proteinase K, mix and incubate at 37°C for 1 hour, during which time it is inverted several times.

[0112] (2) Detection of clinical samples

[0113] According to the on-site lysing method of clinical samples in step (1), 9 nasal swab samples, 1 bovine lung tissue sample, 1 Aerosol samples, 1 blood sample and 1 milk sample were tested for Mycoplasma bovis RPA-LFD, and at the same time, 1 negative sample was taken from each of the above-mentioned diffe...

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Abstract

The invention discloses primers, probes and a kit for rapidly detecting mycoplasma bovis on site. The kit comprises a forward primer sequence shown as SEQ No.1, a reverse primer sequence shown as SEQ No.2, and a probe sequence shown as SEQ No.3, wherein the 5'-terminal of the reverse primer sequence is labeled by biotin; the 5'-terminal of the probe sequence is labeled by FAM, dSpacer is arranged at the position away from the 5'-terminal by 30 basic groups, and the 3'-terminal is blocked by C3-spacer. The primers, the probe assembly and the kit for detecting the mycoplasma bovis RPA-nfo are high in sensitivity and strong in specificity, the mycoplasma bovis DNA of 10 copies/reaction can be detected at the minimum, and the primers and the probes for detecting RPA-nfo respectively have no cross reaction with other mycoplasmas, as well as the bacteria including pasteurella multocida, mannheimia haemolytica, arcanobacterium pyogenes, histophilus somni, and streptococcus pneumoniae.

Description

technical field [0001] The invention relates to the technical field of microbial detection, in particular to a primer, a probe, a kit for rapid on-site detection of Mycoplasma bovis nucleic acid by using a recombinase polymerase amplification-lateral flow chromatography test strip technology and an application thereof. Background technique [0002] Mycoplasma bovis (Mycoplasmabovis, M.bovis) belongs to Mollusca class, Mycoplasma order, Mycoplasma family, Mycoplasma genus, can cause bovine pneumonia, mastitis, arthritis, genital tract inflammation and abortion and other clinical diseases. Mycoplasma bovis was first isolated from the lung of a calf suffering from pneumonia in my country in 2008. Since then, mycoplasma bovis has been reported in most provinces such as Chongqing, Gansu, Ningxia, Guizhou, Shanxi, and Jilin. The mortality rate of mycoplasma bovis pneumonia cases in some cattle farms in my country is about 10%, and the high one can reach 60%, which causes huge econ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/689C12Q1/6844C12Q2521/507C12Q2565/625
Inventor 王洪梅赵贵民侯佩莉丁乃峥何成强何洪彬
Owner SHANDONG NORMAL UNIV
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