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Sampling method and detection method of detecting common vertical transmission pathogen of newborn piglet

A technology of vertical transmission and newborn piglets, which is applied in the field of sampling for detection of common vertically transmitted pathogens in newborn piglets. It can solve problems such as growth inhibition of piglets, economic troubles in the pig industry, and large-scale infection of piglets. It is highly targeted and beneficial. Disease prevention and control and purification, avoiding the effects of cumbersome operations

Pending Publication Date: 2017-09-12
SHANGHAI CHUANG HONG BIOTECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] The vertically transmitted disease of pigs refers to a certain disease suffered by sows. The pathogen can be directly transmitted to piglets through the ovary and placenta. Almost all vertically transmitted pathogens can be transmitted horizontally, causing large-scale and long-term infections in piglets, resulting in The growth and development of piglets are inhibited, or even die, causing troubles and huge economic losses to the pig industry

Method used

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  • Sampling method and detection method of detecting common vertical transmission pathogen of newborn piglet
  • Sampling method and detection method of detecting common vertical transmission pathogen of newborn piglet
  • Sampling method and detection method of detecting common vertical transmission pathogen of newborn piglet

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0113] Newborn piglets were tested for parvovirus, the positive control was porcine parvovirus cultured in cells, and the negative control was double distilled water.

[0114] 1.1 Primer design

[0115] Primers were designed according to the sequence of the conserved region of PPV for amplification, and the amplified fragment was 330bp.

[0116] 5'-AAACGGAGGGACTAGCCAT-3'

[0117] 5'-TGCCATACAGCAGAGA-3'

[0118] 1.2 Disease material processing and nucleic acid extraction

[0119] Take the collected umbilical cord blood samples, specifically take the piglet umbilical cord, cut it in the middle, squeeze the umbilical cord blood into a sterile and dry penicillin bottle, 3-5 drops; at the same time take the afterbirth as the sample to be tested, refer to the virus DNA extraction instructions Extract viral DNA.

[0120] 1.3PCR amplification

[0121] Take 2 μl of the obtained DNA template, 10 μl of 2×Taq mix, 1 μl of PPV upstream and downstream primers, add ddH2O to make the rea...

Embodiment 2

[0130] Newborn piglets were tested for swine fever virus. The positive control was live swine fever vaccine (cell source), which was purchased from Prak Bioengineering Co., Ltd.

[0131] 2.1 Primer design

[0132] Primers were designed according to the CSFV conserved region sequence for amplification, and the amplified fragment was 252bp.

[0133] 5'-AAACGGAGGGACTAGCCAT-3'

[0134] 5'-TGCCATACAGCAGAGA-3'

[0135] 2.2 Disease material processing and nucleic acid extraction

[0136] Take the collected tonsils, cut them into pieces and homogenize them, extract the viral RNA according to the viral DNA / RNA extraction manual, and reverse-transcribe the obtained total RNA to synthesize cDNA according to the reverse transcription kit;

[0137] 2.3PCR amplification

[0138] Take 2 μl of the obtained cDNA template, 10 μl of 2×Taq mix, 1 μl each of CSFV upstream and downstream primers, add ddH2O to make the reaction system 20 μl, mix well and proceed to amplification. The amplifica...

Embodiment 3

[0147] Newborn piglets were tested for PRRSV. The positive control was live porcine reproductive and respiratory syndrome virus CH-1R strain, which was purchased from Shanghai Haili Biopharmaceutical Co., Ltd.; the negative control was double distilled water.

[0148] 3.1 Primer design

[0149] Primers are designed according to the PRRSV genome sequence for amplification, and the target fragment is 340bp (highly pathogenic strain) or 430bp (classical strain).

[0150] 5'-GACCATATGCCAAATAACAACGGC-3'

[0151] 5'-ATTCTCGAGTGCTGAGGGTGATGCTGCTG-3'

[0152] 3.2 Disease material processing and nucleic acid extraction

[0153] Take the collected serum samples, extract viral DNA according to the viral RNA extraction instructions, and reverse-transcribe the obtained total RNA to synthesize cDNA according to the reverse transcription kit;

[0154] 3.3PCR amplification

[0155] Take 2 μl of the obtained cDNA template, 10 μl of 2×Taq mix, 1 μl of PRRSV upstream and downstream primers, ...

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Abstract

The invention provides a sampling method and a detection method of detecting a common vertical transmission pathogen of a newborn piglet. The sampling method comprises the step of sampling the vertical transmission pathogen via afterbirth, cord blood, a tonsil, blood, a nose swab, a throat swab and an anus swab. During detection of a virus, one or more detection samples are processed; viral nucleic acid is extracted; a primer is designed for PCR (polymerase chain reaction) or RT-PCR (reverse transcription-polymerase chain reaction) amplification; agarose gel electrophoresis detection is performed on an amplification product. The sampling method and the detection method can detect the common vertical transmission pathogen of the piglet relatively comprehensively, are simple to operate, short in required lime and high in pertinency, avoid the disadvantages of complex operation, long time consumption, low detection rate and the like of the traditional sampling and detection methods, and facilitate disease prevention and control and purification of the newborn piglet.

Description

technical field [0001] The invention relates to the technical field of molecular diagnosis of animal pathogens, in particular to a sampling method and a detection method for detecting common vertically transmitted pathogens in newborn piglets. Background technique [0002] The vertically transmitted disease of pigs refers to a certain disease suffered by sows. The pathogen can be directly transmitted to piglets through the ovary and placenta. Almost all vertically transmitted pathogens can be transmitted horizontally, causing large-scale and long-term infections in piglets, resulting in The growth and development of piglets are inhibited, and even die, which causes troubles and huge economic losses to the pig industry. Common vertically transmitted pathogens infected by neonatal piglets include swine fever virus (CSFV), porcine parvovirus (PPV), porcine circovirus type 2 (PCV-2), porcine pseudorabies virus (PRV), and porcine reproductive and respiratory syndrome virus. (PRR...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/24C12Q1/70C12Q1/68C12Q1/04C12R1/93C12R1/35C12R1/90C12R1/01
CPCC12Q1/24C12Q1/689C12Q1/6893C12Q1/701C12Q1/705
Inventor 韩相敏马晶晶吴碧清赖志高俊锋张利
Owner SHANGHAI CHUANG HONG BIOTECH
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