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Kit and method for detecting porcine proliferative enteropathy (PPE) pathogenic bacteria Lawsonia Intracellularis

A proliferative and pathogenic bacteria technology, which is applied in the inspection and quarantine field of livestock infectious disease pathogens, can solve the problems of great differences in sensitivity, troublesome electrophoresis, specificity and sensitivity that cannot meet the needs of detection, etc., to achieve strong specificity and reliability, A high degree of automation and the effect of safeguarding national interests

Inactive Publication Date: 2013-07-17
SICHUAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU OF THE PEOPLES REPUBLIC OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Since the bacterium was not successfully cultured in the culture medium, it was not suitable for the growth of chicken embryos [5] , Cell culture isolation and identification are cumbersome and time-consuming
Successively established IFA, IPMA, PCR and fluorescent hybridization histochemical tests, etc. [14-19] There are some disadvantages in the diagnostic methods, the specificity and sensitivity cannot meet the needs of detection, time-consuming and easy to contaminate
For example: IFA and IPMA are similar in specificity. These two methods are not easy to distinguish the types of bacterial antigens, and are mostly suitable for determining the nature of the disease.
Although PCR has high specificity, its sensitivity varies greatly. PCR cannot detect bacteria in the feces of recessively infected pigs or pathogen-carrying pigs, and there are many false positives. Moreover, when PCR is used for large-scale detection, subsequent electrophoresis Very troublesome, not conducive to high-throughput rapid detection

Method used

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  • Kit and method for detecting porcine proliferative enteropathy (PPE) pathogenic bacteria Lawsonia Intracellularis
  • Kit and method for detecting porcine proliferative enteropathy (PPE) pathogenic bacteria Lawsonia Intracellularis
  • Kit and method for detecting porcine proliferative enteropathy (PPE) pathogenic bacteria Lawsonia Intracellularis

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Example 1: Design and Synthesis of Fluorescent Quantitative PCR Primers and Probes

[0036] 1. Design and synthesis of fluorescent quantitative PCR primers and probes

[0037] According to the gene sequence of porcine proliferative bowel disease published by GenBank, a pair of specific primers and a Taqman probe (Taqman-probe) were designed using Primer Express3.0 in the conservative region of the sequence. The amplified target gene fragment is 131bp long. The probe is labeled with the fluorescent reporter 6-FAM (6-Carboxyfluorescein, 5-carboxyfluorescein) at the 5'end, and the fluorescent reporter 6-TAMRA (6-carboxytetramethylrhodamine, 6-Carboxytetramethylrhodamine) labeling can be labelled using conventional methods and equipment in the art. The primers and probes in this example were synthesized and labeled by Shanghai Jikang Biotechnology Co., Ltd. (see Table 1 for the sequence).

[0038] Table 1 Primers and TaqMan probes

[0039]

[0040] 2. Verification of the rationa...

Embodiment 2

[0045] Example 2 Preparation and identification of standard samples

[0046] Use the primers of the present invention to amplify a specific band of about 130 bp from the DNA of Lawsonia suis. According to the instructions of the gel recovery kit, the target fragment is recovered and cloned into the pMD-T Simple vector, and then transformed into DH5α Escherichia coli, pick white colonies grown on selective medium containing ampicillin, culture at 37°C, extract plasmids, perform rapid PCR identification, after purification of positive recombinant plasmids, send them to Shanghai Jikang Bioengineering Co., Ltd. for sequencing , The positive plasmid with the correct sequence is the positive standard for porcine proliferative enteropathy (PPE). Detect its optical density value at 260nm with a nucleic acid protein detector, and then according to Avogadro's constant: 6.02×10 23 Convert to copy number / μL, store at -20°C for later use.

Embodiment 3

[0047] Example 3 Optimization of Fluorescence Quantitative PCR Reaction Conditions

[0048] Using the 25ul system recommended by TaqMan PCR Master Mix (ABI) reagents, using the same concentration of positive plasmid as a template, choosing different concentrations of primers and probes, using the matrix method to optimize the optimal concentration of primers and probes to obtain the lowest reaction The Ct value and the maximum increase in fluorescence intensity (ΔRn) can improve the amplification efficiency and sensitivity of the reaction, and optimize the cycle parameters.

[0049] The optimized reaction conditions of fluorescent quantitative PCR: the matrix method was used to screen out the best primers and probes with final concentrations of 0.6μmol / L and 0.4μmol / L, respectively, and the best annealing extension temperature was 60℃. In summary, the optimal reaction system is: 2×TaqMan PCRMaster Mix 12.5ul, each of the upstream and downstream primers is 0.6μmol / L, the probe is 0....

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Abstract

The invention belongs to the field of pathogenic inspection and quarantine of infectious disease of livestock, and particularly relates to a kit and a method for detecting Lawsonia Intracellularis. In order to realize the purpose of the invention, the technical scheme adopted by the invention is to provide a kit for detecting porcine proliferative enteropathy (PPE) pathogenic bacteria Lawsonia Intracellularis, wherein the kit comprises a primer pair for amplifying DNA (deoxyribonucleic acid) segments of a sample to be detected in the DAN of the extracted sample. According to the invention, through sufficient test screening and optimization, and original creation of a primer probe, reaction conditions, positive control and optimal matching conditions, the method capable of quickly and reliably carrying out PCR (polymerase chain reaction) detection on PPE is established. According to the invention, quantification of a DNA template is realized; the kit and the method have the characteristics of high sensitivity, stronger specificity, stronger reliability, high degree of automation and no pollution, can overcome possible false positive, and are real-time and accurate; and the kit and the method can play important roles in aspects of porcine proliferative enteropathy inapparent infection, can quickly diagnose and monitor ill pigs in real time at early stage and the like.

Description

Technical field [0001] The invention belongs to the field of inspection and quarantine of infectious disease pathogens of livestock, and specifically relates to a detection kit and a detection method for Lawsonia intracellularis Background technique [0002] Porcine Proliferative Enteropathy (PPE), also known as Porcine Proliferative Enteritis (Porcine Proliferative Enteritis), Porcine Ileitis, Porcine Bowl Disease (Porcine Bowl Disease), Porcine Intestinal Adenomatous Disease ( Porcine Intestinal Adenomatosis), Porcine Proliferate Haemorrhage Enteritis (PHE), etc. [1-4] , But it is generally called porcine proliferative enteropathy. The pathogen of the disease is a kind of obligate intracellular parasitic bacteria, called Lawsona Intracellularis (Lawsona Intracellularis, LI), and some scholars called it Rosononia intracellularis [6] , Gebhart et al. think it belongs to DeSulfovibrionaceae (DeSulfovibrionaceae) [4] . [0003] Lawsonia intracellularis mainly parasitizes in the inte...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 陈世界严玉宝余华田绿波胡娟杨苗郭杨
Owner SICHUAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU OF THE PEOPLES REPUBLIC OF CHINA
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