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Multiple PCR rapid detection kit and detection method for pathogen in aquatic products

A technology for detection kits and detection methods, applied in biochemical equipment and methods, measurement/inspection of microorganisms, resistance to vector-borne diseases, etc., can solve the problems of long detection cycle, low sensitivity, cumbersome operation, etc., and achieve improved detection Sensitivity, improved detection efficiency, and cost-saving effects

Inactive Publication Date: 2008-08-06
GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, many countries, including my country, still use traditional bacterial culture and biochemical identification methods for the detection of these pathogenic bacteria. The detection cycle is long, the operation is cumbersome, and the sensitivity is low, and only one pathogenic bacteria can be detected each time. Bacteria, in the face of increasing multiple mixed pollution, the current experimental diagnosis method is seriously lagging behind

Method used

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  • Multiple PCR rapid detection kit and detection method for pathogen in aquatic products

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Embodiment 1

[0026] Example 1: Primer-specific detection

[0027] According to the designed 3 pairs of specific PCR primers, 40 strains of common pathogenic bacteria were detected by PCR, and the orthogonal reaction was used for multiple tests. The negative and positive results are shown in Table 2. The results showed that all 10 strains of Salmonella detected by invA primers amplified the target fragment of 495 bp, and the other 30 strains of non-Salmonella strains did not have any amplified bands; the 4 strains of Vibrio parahaemolyticus detected by toxR primers all amplified the target fragment of 368 bp Fragments, Vibrio cholerae, Vibrio alginolyticus, Vibrio vulnificus and other non-Vibrio strains did not have any amplified bands; 5 strains of Listeria monocytogenes detected by iap primers all amplified 660bp For the target fragment, non-target fragments less than 660bp were amplified from Listeria welseri and Listeria innocua, and there were no amplified bands in Listeria format and ...

Embodiment 2

[0032] Example 2: Detection of Primer Sensitivity

[0033] The culture solution of standard bacterial strain S.typhimurium CMCC50115, V.parahaemolyticus VPL4-90, L.monocytogenes CMCC54002 (plate counting results are respectively: 2.5 × 10 9 cfu / mL, 1.5×10 9 cfu / mL, 4.7×10 9 cfu / mL) after mixing in equal amounts, do 10-fold gradient dilution, and then inoculate each gradient dilution into 90 mL of brain-heart infusion broth culture medium containing 10 g of minced fish meat (containing the mass of dry powder of brain-heart infusion broth medium) 2% NaCl), after culturing for 10 h, DNA was extracted for multiplex PCR detection. The result is shown in Figure 1. The highest detectable dilution concentration was taken as the highest detection sensitivity of the multiplex PCR in the sample. Then the result shows that the highest detectable dilution is 10 -9 , the concentrations of the three bacteria that can be detected are 2.5cfu / mL Salmonella, 1.5cfu / mL Vibrio parahaemolyticu...

Embodiment 3

[0034] Embodiment three, the enrichment comparison of culture medium

[0035] Use respectively common nutrient broth liquid culture liquid, brain heart infusion broth culture liquid and brain heart infusion broth culture liquid containing 2% (dry powder mass content of brain heart infusion broth medium dry powder mass content) NaCl according to the present invention, At the same time, Salmonella, Vibrio parahaemolyticus and Listeria monocytogenes were enriched and cultured, and the enrichment effects of the three culture solutions were compared.

[0036] Salmonella, Vibrio parahaemolyticus and Listeria monocytogenes were added to 5 mL of culture solution, cultured at 37°C for 18 hours, plate counted, the results are shown in Table 3. Result shows, containing 2% (brain heart infusion broth culture medium dry powder mass content) NaCl NaCl brain heart infusion culture solution of the present invention to Salmonella, Vibrio parahaemolyticus and Listeria monocytogenes Bacteria en...

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Abstract

The invention relates to a microorganism detection device and a method for detecting. A multiple PCR rapid detection reagent kit for pathogenic bacteria in aquatic products is characterized in that the kit comprises 10*PCR buffer, 1.0-3.0mmol / L MgC1 2, 240 mu mol / L dNTP each, 60-200nmol / L salmonella primers, 60-200l / Lvibrio parahaemolyticus primers, 200nmol / L Listeria monocytogenes primers and 1.3-3.0 U Taq enzyme. The method has comparatively good specificity, simultaneous detection for salmonella, vibrio parahaemolyticus and Listeria monocytogenes in the water products can be realized conveniently, rapidly, and sensitively, the detection limit for artificially contaminated water products is 10cfu / mL, and the method provides ideal methods for rapidly detecting food-borne pathogenic bacteria of non-polluted water products and is provided with good application prospect.

Description

【Technical field】 [0001] The invention relates to a microorganism detection device and a detection method. 【Background technique】 [0002] my country is a large aquatic product country. With the development of society, people's concern for health and the needs of international trade, the safety of aquatic products has attracted much attention. The safety of aquatic products has a direct impact on the economic development of fishery and even the international trade of aquatic products. Because aquatic products are rich in nutrients, they are highly susceptible to contamination by various microorganisms. Salmonella spp., Vibrio parahaemolyticus and Listeria monocytogenes are common causes Important pathogenic bacteria that pollute aquatic products. In my country's current national health standards, these three bacteria are listed as pathogenic bacteria that must not be detected in pollution-free aquatic products. At present, many countries, including my country, still use trad...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/04C12Q1/68
CPCY02A50/30
Inventor 杨小鹃吴清平张菊梅郭伟鹏
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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