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Primer, probe and kit for rapidly detecting pasteurella mutocida on site

A Pasteurella, multocida technology, applied in the field of microbial detection, to achieve the effect of strong specificity, high sensitivity, and easy to use

Inactive Publication Date: 2017-06-09
SHANDONG NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In summary, for the RPA-LFD detection of Pasteurella multocida, there are no clear ideas and results that can be used for reference, and technical personnel still need to do a lot of in-depth research

Method used

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  • Primer, probe and kit for rapidly detecting pasteurella mutocida on site
  • Primer, probe and kit for rapidly detecting pasteurella mutocida on site
  • Primer, probe and kit for rapidly detecting pasteurella mutocida 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 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 experimental results.

[0065] The present invention designs primers and probes respectively according to the Kmt1 gene (GengBank No.AF016259) specific to Pasteurella multocida, see Table 1 respectively. When designing the primers, the conservation of the Kmt1 gene in the primer design region was first BLASTed on the Genebank data, all of which were 100% matched...

Embodiment 2

[0086] Example 2: Sensitivity, specificity and repeatability investigation of Pasteurella multocida RPA-LFD detection method

[0087] 1. Test method:

[0088] (1) Sensitivity detection of Pasteurella multocida RPA-LFD

[0089] Dilute the positive plasmid standard 10 times to 6.0×10 6 ~6.0×10 0 Copy / μL, use the primers and probes screened in Example 1 of the present invention to carry out RPA-nfo amplification according to the step (3) in the above Example 1, DNA is used as the template, 2 μL respectively, and pEASY-T3 empty load is negative To test the sensitivity of this method.

[0090] (2) Specific detection of Pasteurella multocida RPA-LFD

[0091] The primers and probes obtained by screening in Example 1 of the present invention are respectively effective for capsule type A Pasteurella multocida, capsule B type Pasteurella multocida, capsule D type Pasteurella multocida, capsule Pasteurella multocida type E, Pasteurella multocida type F capsular, Mycoplasma bovis, Ma...

Embodiment 3

[0104] Embodiment 3: The kit that is used for the detection of Pasteurella multocida

[0105] 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.

[0106] 2. Amplification system and detection method:

[0107] The RPA-nfo reaction system is 25 μL:

[0108]

[0109] 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.

[0110] After the reaction, mix 1 μL with 49 μL LFD detection buffer, immerse the LFD vertically in the above mixed buffer, and observe the results within 5 minutes. If the detection band and the control band appear at the same time, the nucleic a...

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Abstract

The invention discloses a combination of primer and probe for rapidly detecting pasteurella multocida on site by RPA-LFD. A forward primer sequence is shown in SEQ ID No. 1; a reverse primer sequence is shown in SEQ ID No. 2; a probe sequence is shown in SEQ ID No. 3. The invention also discloses a kit for detecting pasteurella multocida. The pasteurella multocida RPA-nfo detection combination of primer and probe and kit have high sensitivity and strong specificity, at least can detect six copied / reacted Pasteurella multocida DNAs, and can perform sensitive, specific and rapid detection of Pasteurella multocida DNA on crude lysate of a sample to be detected within 25 min by means of a constant-temperature water bath kettle or human armpit temperature without special instrument and equipment, so as to be suitable for the diagnosis of the pasteurella mutocida disease on site or base.

Description

technical field [0001] The invention relates to the technical field of microbial detection, in particular to primers, probes and kits for on-site rapid detection of Pasteurella multocida by applying recombinase polymerase amplification-lateral flow chromatography test strip technology. Background technique [0002] Pasteurella mutocida (Pm) is a Gram-negative pathogen that causes a variety of pasteurellosis in livestock and poultry. It generally parasitizes the upper respiratory tract of animals and is conditionally pathogenic. Under the condition, it can cause the occurrence of hemorrhagic septicemia or respiratory system disease in carrier animals. Among domestic animals, cattle, pigs, rabbits, and sheep are more frequently affected, and goats, deer, camels, horses, donkeys, dogs, cats, and minks can also be infected. Among poultry, chicken, turkey and duck are most susceptible, followed by geese and pigeon. According to the specificity of capsular antigen, Pm can be div...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/01
CPCC12Q1/6844C12Q1/689C12Q2521/507C12Q2565/625
Inventor 何洪彬赵贵民侯佩莉王洪梅何成强
Owner SHANDONG NORMAL UNIV
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