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Duplex PCR detection kit and detection method for Listeria monocytogenes and Enterococcus faecium

A technology for Listeria and Enterococcus faecium, which is applied in biochemical equipment and methods, microorganism-based methods, and microbial determination/inspection to achieve the effects of convenient identification, strong specificity and high sensitivity

Active Publication Date: 2015-08-19
INNER MONGOLIA AUTONOMOUS REGION ACAD OF AGRI & ANIMAL HUSBANDRY SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Currently, for Listeria monocytogenes and Enterococcus faecium, there is no report of a PCR method that can simultaneously detect these two pathogens

Method used

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  • Duplex PCR detection kit and detection method for Listeria monocytogenes and Enterococcus faecium
  • Duplex PCR detection kit and detection method for Listeria monocytogenes and Enterococcus faecium
  • Duplex PCR detection kit and detection method for Listeria monocytogenes and Enterococcus faecium

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Embodiment 1

[0018] Embodiment 1. Design and screening of primers of the present invention

[0019] According to the known full-length genome sequences of Listeria monocytogenes and Enterococcus faecium, specific genes were screened for primer design, and the hlyA gene of Listeria monocytogenes and the ddl gene of Enterococcus faecium were finally selected, and their specificity was designed and screened. Primers (Table 1), marked as LM-F (SEQ ID NO: 1), LM-R (SEQ ID NO: 2), EFM-F (SEQ ID NO: 3), EFM-R (SEQ ID NO :4).

[0020] Table 1 Primer information

[0021]

Embodiment 2

[0022] Example 2. Establishment and optimization of screening double PCR reaction system

[0023] 2.1 Genome Extraction

[0024] The clinical isolates of Enterococcus faecium and the clinical isolates of Listeria monocytogenes (preserved in our laboratory) were inoculated on nutrient agar medium for pure culture, and then a single colony was picked and cultured in the corresponding enrichment solution until logarithmic growth In the later stage, 2 mL of the bacterial liquid was centrifuged at 10,000 rpm for 1 min to collect the bacterial cells, and the genomic DNA of the two bacterial strains were extracted with a bacterial DNA extraction kit, and stored at -20°C for future use.

[0025] 2.2 Establishment and optimization of multiplex PCR reaction system

[0026] A 25 μL PCR reaction system was used for detection, including: 12.5 μL of 2x Es Taq MasterMix, 2 μL of DNA template, 0.25 μL of each primer, and sterilized deionized water to make up to 25 μL. The reaction condition...

Embodiment 3

[0027] Embodiment 3. The specificity and the sensitivity of double PCR reaction system

[0028] 3.1 Specificity of duplex PCR

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Abstract

The invention relates to a duplex PCR detection kit for Listeria monocytogenes and Enterococcus faecium and a detection method using the kit. The kit detects a primer pair of Listeria monocytogenes hlyA and a primer pair of the dd1 gene of an Enterococcus faecium strain; if specific amplification bands are obtained at 174 bp and 557 bp, Listeria monocytogenes and Enterococcus faecium are positive; and the kit can simultaneously detect Listeria monocytogenes and Enterococcus faecium. The lower limit of the detection sensitivity of the is lower than a minimum infective dose of pathogenic bacteria, which is of great significance to later clinical application and to monitoring of Listeria monocytogenes and Enterococcus faecium.

Description

technical field [0001] The invention relates to a kit for Listeria monocytogenes and Enterococcus faecium, which belongs to the field of animal epidemic molecular biology detection methods and detection reagents, and in particular to a double PCR for synchronously performing Listeria monocytogenes and Enterococcus faecium Detection kit and application kit Biological detection method for dual PCR detection of Listeria monocytogenes and Enterococcus faecium. Background technique [0002] Listeria monocytogenes (Listeria monocytogenes) is a facultative intracellular parasite that can cause zoonotic listeriosis. The animal listeriosis it causes causes pigs, sheep, cattle, rabbits Meningoencephalitis, abortion, and acute sepsis occurred. Enterococci (Enterococci) belong to the normal flora in humans and animals, and are also important opportunistic pathogenic bacteria. Among the cases of infection caused by Enterococcus, the proportion of infection caused by Enterococcus faeciu...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/01
Inventor 赵世华詹树柏杜斌凤英杨斌高娃达赖宝力格陈伟宋爱军
Owner INNER MONGOLIA AUTONOMOUS REGION ACAD OF AGRI & ANIMAL HUSBANDRY SCI
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