Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Primer group using vtaA9 gene as target spot and application of primer group to identification and diagnosis of haemophilus parasuis

A technology of Haemophilus parasuis and primer set, applied in the field of specific primer set, reagent, primer set for identification and diagnosis of Haemophilus parasuis, which can solve the problems of inability to distinguish similar strains, enhanced specificity, and decreased sensitivity And other issues

Inactive Publication Date: 2014-05-14
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
View PDF2 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2001, Oliveria established a classic PCR method based on the 16S rRNA gene, evaluated the effectiveness of this method from the detection of clinical samples, and showed that the sensitivity of this method is higher than that of the traditional bacterial culture isolation method, but it cannot distinguish the same origin from A similar strain of A.indolicus in the upper respiratory tract of pigs, because the similarity of this segment of gene sequence with A.indolicus reaches 97.4-97.7%
In order to overcome this problem, Angen improved the PCR method with 3 specific primers in 2007, and the sensitivity was decreased by 10 times while the specificity was enhanced.
In 2009, Tumi used real-time PCR to detect H.parasuis, but there was still the problem of non-specific reaction with Pasturella mairii strain
However, using the vtaA gene as the only specific gene to distinguish H.parasuis from related strains has not been reported

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Primer group using vtaA9 gene as target spot and application of primer group to identification and diagnosis of haemophilus parasuis
  • Primer group using vtaA9 gene as target spot and application of primer group to identification and diagnosis of haemophilus parasuis
  • Primer group using vtaA9 gene as target spot and application of primer group to identification and diagnosis of haemophilus parasuis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 The establishment of a PCR method for the identification and diagnosis of Haemophilus parasuis with the vtaA9 gene as the target and the sensitivity and specificity analysis

[0028] 1. Sample material

[0029] The samples of the present invention include 15 H.parasuis reference strains, see Table 1, 24 non-H.parasuis strains, and purified genomic DNA of 10 related strains, see Table 2. At the same time, 1.0 g sample was added to 500 μl PBS buffer solution from the clinically suspected H. parasuis infection material for grinding, and then the strains were directly separated and detected by PCR.

[0030] Table 1. H.parasuis reference strains used in the present invention

[0031]

[0032] Table 2 is applied to non-H.parasuis bacterial strains in the present invention

[0033]

[0034] H.parasuis isolated from disease materials were grown in solid TSA (Trypticase Soy Agar) and liquid TSB (Trypticase Soy Broth) medium, supplemented with NAD (10 μg / ml) and...

Embodiment 2

[0058] Example 2 Application of the PCR method with vtaA9 as the target in the identification and diagnosis of Haemophilus parasuis

[0059] In order to further describe the reliability of detecting Haemophilus parasuis clinical samples with vtaA9 as the target of the present invention. The present invention collects 121 clinical samples of 53 pigs infected with Haemophilus parasuis (H.parasuis) from 2008 to 2011. Samples were collected from different tissues and organs, including joint fluid, nasal swabs, heart, lung, and pleural effusion, and the disease materials were immediately sent to the laboratory for bacterial isolation and culture. At the same time, 1.0 g of the sample was added to 500 μl of PBS buffer solution for grinding, and then PCR detection was performed directly.

[0060] 90 and 88 of the 121 samples were positive by vtaA9PCR and 16S rRNA PCR respectively, the positive ratios were 74.4% and 72%, respectively, and 83 of the 121 samples were positive for bacte...

Embodiment 3

[0064] Example 3 Identification and typing of clinical isolates obtained by vtaA9 gene isolation

[0065]In Example 2, the vtaA9PCR method was used to detect and isolate 90 H.parasuis clinical isolates. They had typical H.parasuis characteristics. The biochemical reaction revealed that 90 clinical isolates were NAD-dependent and had no hemolysis. The use of urease is negative, and the positive of catalase is consistent with the results of Kielstein et al. (2001). The 90 H.parasuis clinical isolates showed different isolation sites and different isolation rates, the isolation rate of lung (n=34; 37.8%), the isolation rate of pleural effusion (n=14; 15.6%), the isolation rate of heart (n =13;14.4%), joint fluid separation rate (n=10;11.1%), tracheal separation rate (n=8,8.9%), brain separation rate (n=6;6.7%), nasal separation rate (n =4; 4.4%), tonsil separation rate (n=1; 1.1%). Similar to the results of Nedbalcova (2006), the success rate of separation and culture of pleura...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a primer group using a vtaA9 gene as a target spot and application of the primer group to identification and diagnosis of haemophilus parasuis. Researches find that a PCR (Polymerase Chain Reaction) method using the vtaA9 gene as the target spot is capable of accurately distinguishing different pathogenicities, and can simultaneously settle in swine upper respiratory tract H.parasuis and similar strains A.indolicus, A.minor and A.porcinus thereof. In addition, based on the vtaA9 gene sequence, the invention designs and synthesizes a specific primer, wherein nucleotide sequences of the primer are shown as SEQ ID NO.1 and SEQ ID NO.2. Specificity experiments indicate that according to the method disclosed by the invention, the H.parasuis and the A.indolicus as well as the A.minor and the A.porcinus can be completely distinguished. Sensitivity experiments indicate that by using the method disclosed by the invention, genomes DNA of not less than 20CFU / ml and 5pg / mu l can be detected. In addition, a positive result of the PCR method using the vtaA9 as the target spot for a clinical sample is higher than that of isolated culture of a strain and a classic 16S rRNA (ribosomal ribonucleic acid) PCR method. The invention provides a novel method for differential diagnosis of haemophilus parasuis and epidemiological monitoring.

Description

technical field [0001] The present invention relates to a new target for identifying and diagnosing Haemophilus parasuis and a specific primer set based on the target, in particular to a target of vtaA9 gene for identifying and diagnosing Haemophilus parasuis A primer set for Haemophilus parasuis and its application in the preparation of reagents for identifying and diagnosing Haemophilus parasuis. The invention belongs to the detection field of Haemophilus parasuis. Background technique [0002] Haemophilus parasuis is a member of NAD (Nicotinamide Adenine Dinucleotide)-dependent Pasteurellaceae family, which can cause pig Disease characterized by systemic symptoms such as serositis, peritonitis, and meningitis. The diagnosis of the disease usually relies on the comprehensive diagnosis of clinical symptoms, autopsy changes and pathogenic bacteria isolation. Due to clinical mixed infection and the treatment of antibiotics, the clinical symptoms are complicated, and the d...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/04C12Q1/686C12Q2531/113
Inventor 张艳禾
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com