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

Common primer nucleic acid amplification method for detecting three pig viruses synchronously and kit

A technology of simultaneous detection and shared primers, applied in the biological field, can solve the problems of harm to the pig industry and the difficulty of disease prevention and control.

Active Publication Date: 2014-04-30
INSPECTION & QUARANTINE TECH CENT OF GUANGDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
View PDF3 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] The above three porcine viruses are important pathogens that cause reproductive disorders in pigs. The diseases caused by them are common in the pig industry and are difficult to control. Clinically, it has been found that 2 or even 3 kinds of porcine viruses are mixed infection, which has caused serious harm to the pig industry.

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
  • Common primer nucleic acid amplification method for detecting three pig viruses synchronously and kit
  • Common primer nucleic acid amplification method for detecting three pig viruses synchronously and kit
  • Common primer nucleic acid amplification method for detecting three pig viruses synchronously and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0103] Embodiment 1 Simultaneously detects the new method of nucleic acid amplification with common primers of three kinds of porcine viruses

[0104] A novel nucleic acid amplification non-diagnostic method for simultaneous detection of PCV-2, PRV and PPV three porcine viruses, the method comprising:

[0105] (1) Extract viral nucleic acid from the sample;

[0106] (2) Perform PCR amplification on the extracted nucleic acid, wherein

[0107] The PCR amplification reaction system uses a 40 μL reaction system:

[0108] The consensus primers are as shown in SEQ ID No.1 and SEQ ID No.2, wherein the bases at the 4th, 8th, and 11th positions from the 5' end of the primer SEQ ID No.1 are modified by locked nucleic acid (LNA). The 4th, 7th, and 10th bases from the 5' end of SEQ ID No.2 are modified with locked nucleic acid (LNA), and the final concentration is 250nmol / L; the specific primer for detecting PRV has a sequence such as SEQ ID No.5 And SEQ ID No.6, its primer final conc...

Embodiment 2

[0122] Embodiment 2 The inventive method specificity, sensitivity detection

[0123] 1. Specificity test

[0124] Using PCV-2, PRV, PPV cells to proliferate virus strains and virus vaccines, after extracting and purifying viral nucleic acids, adopt the reaction system and amplification reaction conditions determined in Example 1, to single viral nucleic acid templates and templates mixed with three viral nucleic acids The detection test was carried out, and the results were as follows figure 2 show, figure 2 Middle M: ​​standard molecular weight marker of nucleic acid; 1: detection results of mixed samples of equal amounts of cytotoxic nucleic acid of PCV-2, PPV and PRV; 2: equal amount of viral nucleic acid extracted from commercial vaccine products of the three viruses 3: Detection results of PPV cytotoxic nucleic acid samples; 4: Detection results of PCV-2 cytotoxic nucleic acid samples; 5: Detection results of PRV cytotoxic nucleic acid samples; 6: Negative control. D...

Embodiment 3

[0138] Example 3 Detection of Clinical Samples and Artificially Prepared Poisoned Pig Semen

[0139] 1. Extract and purify viral nucleic acid from samples

[0140] The method of the invention can be used to detect the virus in clinical samples such as animal serum, semen and tissue, and the virus in cell culture fluid. After the sample is pretreated by grinding and centrifugation, the viral nucleic acid can be extracted and purified by TRIzol extraction method or commercial silica gel column extraction method.

[0141] (1) Sample pretreatment

[0142] 1) Pretreatment of porcine tissue samples: take an appropriate amount of porcine viscera, muscle and other tissue samples, cut them into pieces and grind them, add sterilized PBS buffer at a ratio of 1:5 (W / V), transfer them into a centrifuge tube, shake and mix well, Freeze and thaw three times, centrifuge at 5000g for 1 min, and take the supernatant for subsequent nucleic acid extraction and purification.

[0143] 2) Pretrea...

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 provides a novel nucleic acid amplification detection method for synchronously and rapidly detecting three important pig pathogeny of porcine circovirus type 2, porcine pseudorabies virus and porcine parvovirus by LNA (Locked nucleic acid) modified base common primer guidance as well as a detection kit. A pair of common primers containing LNA modified bases and three pairs of 5'end virus specific primers with common primer base sequences are designed and screened, and the specific PCR (Polymerase Chain Reaction) amplification reaction is guided mainly by the LNA modified base common primers in the reaction process by an optimized reaction system and two steps of PCR amplification reaction conditions, so that the porcine circovirus type 2, porcine pseudorabies virus and porcine parvovirus are detected simply, conveniently and synchronously. The novel nucleic acid amplification detection method and the kit which are provided by the invention verify that the LNA modified base common primers can efficiently guide multiple PCR amplification, and provide research and experiment basis for the research and the invention of the modified base common primers applicable to multiple PCR systems with other functions.

Description

technical field [0001] The invention belongs to the field of biological technology, and specifically, provides a novel nucleic acid amplification detection method and a detection kit for synchronously detecting porcine circovirus type 2, porcine pseudorabies virus and porcine parvovirus. Background technique [0002] (1) Status quo of simultaneous and rapid detection technology for swine pathogens [0003] Pigs can be infected with a variety of pathogens, and pigs have similar or even clinically difficult to distinguish symptoms after a variety of diseases, which brings difficulties to the accurate diagnosis of diseases. Scientists from all over the world are working hard to research and develop methods that can simultaneously detect and identify pathogens of various diseases. The rapid development of molecular biology technology provides a technical basis for the simultaneous identification and detection of various swine diseases. [0004] In swine disease detection, the m...

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
Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/686C12Q1/70C12Q2537/143C12Q2525/117C12Q2531/113
Inventor 陈茹高小博宋长绪薛春宜于晓璐段燕喻李艳刘志玲陈芳
Owner INSPECTION & QUARANTINE TECH CENT OF GUANGDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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