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Staphylococcus aureus multiple PCR-MIX rapid detection kit and detection method thereof

A technology for detection kits and staphylococci, which is applied in the direction of microbe-based methods, microbiological determination/inspection, biochemical equipment and methods, etc. It can solve the problems of gene deletion, low specificity of primers, false positives, etc., and achieve cycle time Short, sensitive, and specific effects

Active Publication Date: 2009-12-30
GUANGDONG INST OF MICROORGANISM +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of these methods focus on drug resistance-associated genes and toxin genes, and the absence of genes and low specificity of primers are likely to cause false positives

Method used

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  • Staphylococcus aureus multiple PCR-MIX rapid detection kit and detection method thereof
  • Staphylococcus aureus multiple PCR-MIX rapid detection kit and detection method thereof
  • Staphylococcus aureus multiple PCR-MIX rapid detection kit and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1: the establishment of multiplex PCR-MIX rapid detection kit of Staphylococcus aureus

[0023] 1. Establishment of Multiplex PCR Reaction System

[0024] 1. Primer design

[0025] Consult the references, determine the relevant specific genes to be studied in the experiment, and select more than 3 genes as the research objects. Log in to the NCBI website (www.ncbi.nlm.nih.gov), obtain the accession number and nucleotide sequence of the specific gene of the pathogenic bacteria in the GeneBank database, and conduct gene informatics analysis. After analysis, this study chose nuc, clfA, SmaI three specific identification genes.

[0026] Primers were designed and analyzed using Primer Premier 5.0 software to understand the kinetic characteristics of these primers, including primer length, Tm value, secondary structure, mismatch between upstream and downstream primers, stability at the 3′ end, GC content, and length of PCR products, etc. , choose primer pairs wi...

Embodiment 2

[0072] Example 2 Multiplex PCR-MIX Rapid Detection Kit Detects Market Pork Samples

[0073] 1. Kit composition

[0074] One PCR MIX (600 μ l, the specific composition is as described in Example 1, the difference is that the concentrations of SEQ NO.1 and 2, SEQ NO.3 and 4 and SEQ NO.5 and 6 are respectively 550nmol / L), one Negative control DNA (30 μl), one positive control DNA (30 μl), one sterile water (1000 μl).

[0075] 2. Sample testing process

[0076] (1) Enrichment culture

[0077] 10 g was randomly sampled by aseptic operation, added to 90 mL of broth containing 7.5% NaCl, and incubated at 37° C. for 10 hours.

[0078] (2) DNA extraction

[0079] 1) 1.5mL sample, 12000r / min for 2min, discard the supernatant; 2) suspend the precipitate in 200μL acetone, shake and mix, ice bath for 5min, 10000r / min, 2min, discard the supernatant; 3) dissolve the precipitate in 500μL TE buffer 4) Resuspend the pellet in 400 μL lysate, pipette repeatedly, keep in ice bath for 5 min, a...

Embodiment 3

[0085] Example 3 Multiplex PCR-MIX Rapid Detection Kit Detects Cake Samples

[0086] 1. Kit composition

[0087] One PCR MIX (600 μl specific composition is as described in Example 1, the difference is that the concentrations of SEQ NO.1 and 2, SEQ NO.3 and 4 and SEQ NO.5 and 6 are respectively 300nmol / L), one negative Control DNA (30 μl), one positive control DNA (30 μl), one sterile water (1000 μl). Participate in Example 1.

[0088] 2. Sample testing process

[0089] (1) Enrichment culture

[0090] 10 g was randomly sampled by aseptic operation, added to 90 mL of broth containing 7.5% NaCl, and incubated at 37° C. for 10 hours.

[0091] (2) DNA extraction

[0092] 1) 1.5mL sample, 12000r / min for 2min, discard the supernatant; 2) suspend the precipitate in 200μL acetone, shake and mix, ice bath for 5min, 10000r / min, 2min, discard the supernatant; 3) dissolve the precipitate in 500μL TE buffer 4) Resuspend the pellet in 400 μL lysate, pipette repeatedly, keep in ice bat...

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Abstract

The invention discloses a staphylococcus aureus multiple PCR-MIX rapid detection kit and a detection method thereof; the detection kit comprises PCR MIX; wherein, the PCR MIX contains 2*PCR buffer, 1.0-3.0mmol / L of MgCl2, 180-220mu mol / L of dNTP each, heat-resisting deoxyribonuclease nuc gene primer of which the base sequence is SEQ NO.1 and the concentration of SEQ NO.1 is 100-600nmol / L, plasma-coagulase clfA gene primer of which the base sequence is SEQ NO.3 and the concentration of SEQ NO.4 is 100-600nmol / L, SmaI restriction fragment specific sequence primer of which the base sequence is SEQ NO.5 and the concentration of SEQ NO.6 is 100-600nmol / L, 20-60U of Taq enzyme and bromophenyl blue dye. The multiple PCR-MIX rapid detection kit and the detection method provided by the invention have simple configuration, the detection kit and the detection method can be used in industrialized production; the detection method is sensitive and has short detection cycle and strong maneuverability, so that the method can be widely applied in the fields of food hygiene, environmental monitoring, etc.

Description

Technical field [0001] The invention belongs to the field of food microbial safety monitoring, and relates to a method for rapid detection of microorganisms, which uses specific genes of microorganisms and multiplex PCR technology to rapidly detect and identify microorganisms. Specifically, it relates to a multiplex PCR-MIX rapid detection kit for Staphylococcus aureus. and detection methods. Background technique [0002] "Food is the first necessity of the people", food is the material basis for human survival, and food safety is a major issue related to human health and social development. In recent years, domestic and foreign food safety incidents have continued to occur, especially Staphylococcus aureus, Salmonella, Escherichia coli O157, Listeria monocytogenes, Foodborne diseases caused by Vibrio parahaemolyticus and Bacillus cereus are the most prominent. According to statistics, foodborne diseases caused by microorganisms account for more than 50% of the total in bo...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12R1/445
Inventor 徐晓可吴清平邓梅清张菊梅郭伟鹏
Owner GUANGDONG INST OF MICROORGANISM
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