Taqman-MGB fluorescent quantitative PCR kit and method for detecting 12 common viruses and bacteria of pig at same time

A pcv-2-taqman-mgb, fluorescence quantitative technology, used in biochemical equipment and methods, microbial determination/inspection, etc.

Active Publication Date: 2016-06-01
BEIJING YISEN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no patent or literature on using Taqman-MGB probe to simultaneous...

Method used

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  • Taqman-MGB fluorescent quantitative PCR kit and method for detecting 12 common viruses and bacteria of pig at same time
  • Taqman-MGB fluorescent quantitative PCR kit and method for detecting 12 common viruses and bacteria of pig at same time
  • Taqman-MGB fluorescent quantitative PCR kit and method for detecting 12 common viruses and bacteria of pig at same time

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0138] Example 1 Establishment of simultaneous detection of 12 kinds of pig common viruses and bacteria Taqman-MGB fluorescent quantitative PCR detection method

[0139] 1. Design primers and Taqman-MGB probes

[0140] According to the specific sequences of 12 kinds of pathogens, the sequences were compared by DNAstar software to find out the specific conserved sequences of various pathogenic gene sequences. Use PrimerPrimer5.0 software to design primers and probes. Multiple pairs of specific primers and probes were obtained, and after comparison and screening, a set of optimal primers and MGB probes were finally determined.

[0141] Table 112 kinds of pathogenic primer probe sequence list

[0142]

[0143]

[0144] Compared with the traditional TaqMan-TAMRA probe, the TaqMan-MGB probe has the following advantages: (1) Increase the TM value - the average 15bases can increase 18°C, which can shorten the length of the probe, especially for sequences with high AT conten...

Embodiment 2

[0190] Example 2: Preparation of Taqman-MGB Fluorescent Quantitative PCR Kit for Porcine Common Viruses and Bacteria and Sample Detection

[0191] 1. Preparation of the kit:

[0192] Reagent 1 preparation:

[0193] Table 7 Fluorescent quantitative PCR reaction solution A configuration table (180μl)

[0194] Reagent

Usage amount

PCV-2-F

4μl

PCV-2-R

4μl

PCV-2-Taqman-MGB

4μl

PRV-F

2μl

PRV-R

2μl

PRV-Taqman-MGB

2μl

[0195] PPV-F

4μl

PPV-R

4μl

PPV-Taqman-MGB

4μl

SS-II-F

4μl

SS-II-R

4μl

SS-II-Taqman-MGB

4μl

2×One Step RT-PCR Buffer

100μl

Sterile deionized water

38μl

[0196] Table 8 Fluorescence quantitative PCR reaction solution B configuration table (180μl)

[0197] Reagent

Usage amount

CSFV-F

8μl

CSFV-R

8μl

CSFV-Taqman-MGB

8μl

FMDV-F

2μl...

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Abstract

The invention provides a Taqman-MGB fluorescent quantitative PCR kit and a method for detecting 12 common viruses and bacteria of pigs at the same time. The kit comprises PCR reaction liquids A/B/C, wherein the PCR liquids comprise primer pairs and Taqman probes for porcine parvovirus (PPV), type-II streptococcus suis (SS-II), a porcine pseudorabies virus (PRV), type-II porcine circovirus (PCV-2), a hog cholera virus (CSFV), a pig foot and mouth disease virus (FMDV), a porcine reproductive and respiratory syndrome virus (PRRSV), a high pathogenicity porcine reproductive and respiratory syndrome virus strain (Hp-PRRSV), a transmissible gastroenteritis virus (TGEV), an epidemic diarrhea virus (PEDV), rotavirus (PRTV) and a swine influenza virus (SIV) respectively. 12 pathogens of pigs can be detected rapidly and effectively at the same time, the detection method is high in accuracy, specificity and sensitivity and is good in stability, and rapid diagnosis and effective detection on pathogens to be detected can be achieved.

Description

technical field [0001] The invention relates to the field of molecular biology detection of bacteria and viruses, in particular to a Taqman-MGB fluorescent quantitative PCR kit and method for simultaneously detecting 12 kinds of pig common viruses and bacteria. Background technique [0002] my country is a large agricultural and animal husbandry country. Pig farming is mainly large-scale and intensive farming. The high-density breeding method also makes it difficult to prevent infectious diseases in pigs, and the problem is becoming more and more significant. Moreover, multiple pathogens are infected at the same time, which makes diagnosis and prevention of diseases very difficult. [0003] Porcine parvovirus (PPV), Streptococcus suis type 2 (SS-II), porcine pseudorabies virus (Porcinepseudorabies virus, PRV), porcine circovirus type II (Porcine circovirus- 2, PCV-2), transmissible gastroenteritis virus (Transmissiblegastroenteritisvirus, TEGV), epidemic diarrhea virus (Por...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12Q1/04
Inventor 王新杰高姗姗孙晓明胡祥钰
Owner BEIJING YISEN BIOTECH
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