A method for rapid detection and identification of Listeria bacteria

A technology for Listeria monocytogenes and Listeria monocytogenes, which is applied in the field of molecular biological detection, can solve the problems of high cost, complicated steps, time, and high work intensity, and achieve the effects of improving efficiency, good application prospects, and saving time

CN104342496BActive Publication Date: 2016-10-05SHANGHAI OCEAN UNIV
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Patents(China)
Current Assignee / Owner
Publication Date
2016-10-05

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Abstract

The invention belongs to the technical field of molecular biological detection, and provides a method for detecting and identifying six pairs of PCR (polymerase chain reaction) primers of six kinds of Listeria bacteria from an environment respectively. The PCR primer pairs are designed according to a specific target gene iap (inhibitor of apoptosis protein) of the Listeria bacteria, the target gene is high in stability and specificity, and the six kinds of Listeria bacteria existing in the environment can be rapidly and accurately identified by virtue of a reasonable PCR system and a gel electrophoresis detection means. The detection method comprises the following steps of (1) extracting a sample DNA (deoxyribonucleic acid), and performing PCR amplification by adopting different primer pairs respectively; (2) judging whether a sample to be detected contains the Listeria bacteria or not according to the length of a PCR amplification product fragment. According to the method, the six kinds of Listeria bacteria in the environment and a food sample can be rapidly, accurately and sensitively detected and identified, so that the efficiency is improved, time is saved, and moreover, the detection cost is lowered.
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Description

technical field

[0001] The invention relates to a method for rapidly detecting and identifying bacteria of the genus Listeria in the environment, belonging to the technical field of molecular biological detection. Background technique

[0002] Listeria spp. Firmicutes, along with Bacillus, Clostridium, Enterococcus, Streptococcus and Staphylococcus; low G+C content (36-42%), aerobic or Facultative anaerobic, asporotic bacteria that produce flagella at 10-25°C and are capable of motility. Currently, there are 6 species of Listeria, namely Listeria monocytogenes (L.monocytogenes), Listeria ovis (L.ivanovii) (L. innocua), L. welshimeri, L. seeligeri, and L. grayi. These six species are widely distributed in the environment, and Listeria monocytogenes and Listeria ovis are pathogenic. Listeria monocytogenes is a zoonotic pathogen that can cause severe septicemia and meningitis, and cause listeriosis with a mortality rate of about 20-30% in humans; food is the main route of hu...

Examples

Embodiment 1

[0046] Example 1 PCR detection method for specificity evaluation of different strains

[0047] 1 Materials and methods

[0048] 1.1 Materials

[0049] 1.1.1 Medium

[0050] Listeria Selective Enrichment Solution (LB1, LB2), Tryptone Soy Broth (TSB), Tryptone Soy Agar (TSA) were purchased from Beijing Land Bridge Technology Co., Ltd.; Listeria Selective Agar (PALCAM), Brain heart infusion (BHI), antibiotics, etc. were purchased from OXOID, UK;

[0051] 1.1.2 Main reagents

[0052] PCR Premix (including rTaq, 10×buffer(+Mg 2+ ), and dNTP Mixture), DNA Marker (DL2000bp), and DNA Recovery Kit were purchased from Dalian Bao Biological Engineering Co., Ltd.; Biospin Bacterial Genomic DNA Extraction Kit was purchased from Hangzhou Bioer Company; agarose, EB (ethidium bromide) Bought from Tiangen Biotechnology Company; biochemical identification kits were purchased from Beijing Land Bridge Co., Ltd.; other reagents: anhydrous ethanol, 95% ethanol, glycerin, sodium chloride, etc. ...

Embodiment 2

[0101] Example 2 The experiment of detecting Listeria in the environment represented by fresh pork samples