Primers, probes and kit for rapidly detecting mycobacterium paratuberculosis on site

A technology for mycobacteria and paratuberculosis, applied in the field of microbial detection, to achieve the effects of convenient use, high sensitivity and strong specificity

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

[0009] In summary, for the RPA-LFD detection of Mycobacterium paratuberculosis, there are no clear ideas ...

Method used

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  • Primers, probes and kit for rapidly detecting mycobacterium paratuberculosis on site
  • Primers, probes and kit for rapidly detecting mycobacterium paratuberculosis on site
  • Primers, probes and kit for rapidly detecting mycobacterium paratuberculosis on site

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1: Design and screening of primers and probes

[0063] 1. Design of primers and probes

[0064] At present, there are no specific operating rules for the design of RPA-nfo primers and probes. 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 experimental results.

[0065] The present invention designs forward primers, reverse primers and probes according to the specific IS900 gene (GengBank No. S74401) of Mycobacterium paratuberculosis, as shown in Table 1 respectively. There are 6 sets of RPA combinations of primers and probes. When designing the primers, the conservation of the IS900 gene in...

Embodiment 3

[0107] Embodiment 3: the kit that is used for the detection of Mycobacterium paratuberculosis

[0108] 1. The composition of kit: the primer and probe combination of embodiment 1 screening, positive quality control standard substance, negative quality control standard substance, rehydration buffer, magnesium acetate (280mM), ddH 2 O and lateral flow chromatography test strips.

[0109] 2. Amplification system and detection method:

[0110] The RPA-nfo reaction system is 25 μL:

[0111]

[0112] Add 23.75 μL of the above mixture into the RPA-nfo reaction tube, mix well and dissolve, and finally add 1.25 μL of 280 mM magnesium acetate solution, mix upside down and directly place it in a 37°C constant temperature water bath for 25 minutes.

[0113] After the reaction, mix 1 μL with 49 μL LFD detection buffer, immerse the LFD vertically in the above mixed buffer, observe the result within 5 minutes, and interpret it through the display of the test strip. If the detection ban...

Embodiment 4

[0114] Embodiment 4: the application of RPA-LFD detection method of Mycobacterium paratuberculosis

[0115] 1. Experimental steps

[0116] (1) On-site preparation of clinical samples

[0117] Use 200μL of TE buffer [1.0M Tris-HCl (pH8.0) 10mL, 0.5M Na 2 EDTA·2H 2 O (pH8.0) 2mL, add distilled water to 1000mL] resuspend, blood, serum and other liquid samples directly take 200μL, then add 30μL 10% (w / v) SDS and 3μL 2% (w / v) proteinase K, mix Then incubate at 37°C for 1 hour, during which time it was turned upside down several times.

[0118] (2) Detection of clinical samples

[0119] According to the method of on-site cracking and processing of clinical samples in step (1), 9 stool samples, 1 serum sample, and 4 blood samples that have been amplified with Mycobacterium paratuberculosis-specific PCR primers and sequenced correctly are stored in our laboratory. Samples and 1 milk sample were tested for Mycobacterium paratuberculosis RPA-LFD. At the same time, 1 negative sample...

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Abstract

The invention discloses primers and a probe assembly for rapidly detecting mycobacterium paratuberculosis on site. A forward primer sequence is shown as SEQ ID No.1, a reverse primer sequence is shown as SEQ ID No.2, and a probe sequence is shown as SEQ ID No.3. The invention further discloses a kit for detecting the mycobacterium paratuberculosis. The primers, the probe assembly and the kit for detecting mycobacterium paratuberculosis RPA-nfo are high in sensitivity and strong in specificity, and mycobacterium paratuberculosis DNA of 8 copies/reaction can be detected at the minimum. The special instruments and equipment are not needed, by virtue of a thermostat water bath kettle or the temperature of the human armpit, the sensitive, specific and rapid detection for the mycobacterium paratuberculosis DNA can be carried out on the coarse lysate of a to-be-detected sample within 25min, and the primers, the probes and the kit are suitable for the on-site or primary-level diagnosis work of paratuberculosis.

Description

technical field [0001] The invention relates to the technical field of microbial detection, in particular to a primer, a probe and a kit for rapid on-site detection of Mycobacterium paratuberculosis by applying the recombinase polymerase amplification-lateral flow chromatography test strip technology. Background technique [0002] Paratuberculosis, also known as paratuberculosis enteritis, is a chronic infectious disease caused by Mycobacterium paratuberculosis (MAP) in cattle, sheep, deer, camels and other animals. OIE lists it as a B-class disease, and my country lists it as a second-class animal disease shared by many animals. Mycobacterium paratuberculosis mainly infects cattle, sheep, and deer. Diseased animals and latent infected persons are the main sources of infection, and they can excrete a large amount of pathogenic bacteria through milk, feces, and urine. The prevalence of the disease is characterized by slow development, low morbidity and high fatality rate, an...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/6844C12Q1/689C12Q2521/507C12Q2565/625
Inventor 何洪彬赵贵民王洪梅侯佩莉
Owner SHANDONG NORMAL UNIV
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