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Reagent kit for inspecting active mononucleosis Lee's bacterium RT-PCR and its inspection

A monocytogenes and Listeria technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of high price, low detection limit, complex technology, etc., and achieve easy industrial production. , Accurate detection and identification, the effect of simple configuration method

Inactive Publication Date: 2007-06-20
GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The whole process of the traditional biochemical detection method for Listeria monocytogenes needs at least 4-7 days, and the detection limit is low (only 10 4 cfu / ml(g), time-consuming and labor-intensive, cannot meet the needs of timely, rapid and sensitive detection of pathogenic bacteria in food, and is not conducive to the treatment of food poisoning emergencies
The immunization method is relatively fast, but it is difficult to prepare monoclonal antibodies, which are prone to cross-reaction and poor specificity; the nucleic acid hybridization method is fast and specific, but the sensitivity is low, and it takes 10 3 -10 4 Only cfu / g can get the hybridization signal; the PCR method is fast, specific and highly sensitive, but the results obtained by the PCR method include live bacteria and dead bacteria, which are prone to false positives
So far, there has been no research on solving false positives in China. Although there are a small number of reports abroad on the detection of live Listeria monocytogenes by RT-PCR method, the results are displayed by southern hybridization, which takes a long time to detect, complicated technology, and expensive. It is not easy to popularize and apply, and there is no detailed research on the practical application of sample detection

Method used

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  • Reagent kit for inspecting active mononucleosis Lee's bacterium RT-PCR and its inspection

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1: Listeria monocytogenes RT-PCR living body rapid detection kit

[0031] The detection kit consists of RT system reagents and PCR system reagents.

[0032] RT system reagents include the following reagents: 5×RT-PCR buffer 40 μL, 10 U / uL Rnasin 20 μL, 10 mmol / L dNTP 20 μL, 100 U / μL MLV reverse transcriptase 20 μL, 10 μmol / L upstream primer 40 μL, 10 μmol / L downstream primer 40 μL.

[0033] The PCR system reagents include the following reagents: 10mmol / L dNTP 20μL, 10×PCR reaction buffer 50μL, 2.5U / μL Taq enzyme 10μL, 10umol / L upstream primer 10μL, 10umol / L downstream primer 10μL.

[0034] The upstream primer P1 used in this kit is: 5'-CCT AAG ACG CCA ATC GAA AAG AAA-3', and the downstream primer P2 is: 5'-TAG TTC TAC ATC ACC TGA GAC AGA-3'.

Embodiment 2

[0035]Embodiment 2: RT-PCR live rapid detection method of Listeria monocytogenes in pork

[0036] 1. Sample pretreatment. Take 25g of the positive food sample pork confirmed by the traditional method, cut it into pieces by aseptic operation, put it in a 225mL TSB Erlenmeyer flask, culture it at 37°C and 200r / min for 12h, and filter it out with a funnel with filter paper leftover food.

[0037] 2. RNA extraction

[0038] 1) Take 1 mL of cultured filtrate, after centrifugation, add lysozyme to act at 37°C for 1 h, add 1 ml of TRNzol reagent, mix repeatedly with a pipette several times, and let stand for 5 min;

[0039] 2) Add 200uL chloroform, shake vigorously for 15S, and then let stand for 2-3min;

[0040] 3) 4°C, 12000g, centrifuge for 15min, discard the upper liquid;

[0041] 4) Add 500uL isopropanol, mix thoroughly, and place at room temperature for 10 minutes;

[0042] 5) 4°C, 12000g, centrifuge for 10min, discard the supernatant again;

[0043] 6) Add 75% ethanol, s...

Embodiment 3

[0057] Embodiment 3: RT-PCR live rapid detection method of Listeria monocytogenes in milk

[0058] 1. Sample pretreatment

[0059] Take 25ml of the positive food sample milk confirmed by the traditional method, place it in a 225mL TSB Erlenmeyer flask by aseptic operation, 37°C, 200r / min shaking culture for 16h, use a funnel with filter paper to filter out the milk impurities.

[0060] 2. RNA extraction

[0061] 1) Take 1 mL of cultured filtrate, after centrifugation, add lysozyme to act at 37°C for 1 h, add 1 ml of TRNzol reagent, mix repeatedly with a pipette several times, and let stand for 5 min;

[0062] 2) Add 200 μL of chloroform, shake vigorously for 15 seconds, and then let stand for 2-3 minutes;

[0063] 3) 4°C, 12000g, centrifuge for 15min, discard the upper liquid;

[0064] 4) Add 500uL isopropanol, mix thoroughly, and place at room temperature for 10 minutes;

[0065] 5) 4°C, 12000g, centrifuge for 10min, discard the supernatant again;

[0066] 6) Add 75% eth...

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Abstract

Vital mononucleosis listeria RT-PCR fast inspecting reagent kit and its method are disclosed. The process is carried out by taking mRNA of mononucleosis listeriolysin gene hlyA as target, extracting sample RNA, RT-PCR amplification reacting and electrophoresis inspecting. It is fast, has better sensitivity and operability. It can be used in environmental inspection, food production and commodity quarantine.

Description

[Affiliated technical field] [0001] The invention relates to a rapid detection kit and detection method for detecting live Listeria monocytogenes by using molecular technology, in particular to RT-PCR for active Listeria monocytogenes based on mRNA for the hemolysin gene hlyA A rapid detection kit and a detection method belong to the field of biotechnology. [Background technique] [0002] Listeria monocytogenes (Listeria monocytogenes) (hereinafter referred to as "Listeria monocytogenes") is an important zoonotic pathogen, which can cause meningitis, sepsis and abortion in pregnant women and animals. The mortality rate was extremely high. Listeria monocytogenes widely exists in soil, animals, aquatic products, etc., and is mainly transmitted through food such as milk and dairy products, vegetables, aquatic products, and meat products. The bacterium has been listed as one of the four major food pathogens in the 1990s, and has become a must-check item for food hygiene in man...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 吴清平李善志张菊梅郭伟鹏杨宁
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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