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Fluorescent polymerase chain reaction (PCR) primer, probe and method for detecting A, C and G group of hemolytic streptococcus

A hemolytic streptococcus and probe technology, which is applied in the fields of probes, detection of the whole group of hemolytic streptococci, and fluorescent PCR primers, can solve the problems of high experience requirements for inspectors, cumbersome detection steps, long detection cycle, etc., and achieve The effect of wide detection range, short detection time and large detection amount

Inactive Publication Date: 2012-12-26
SHENZHEN ACAD OF METROLOGY & QUALITY INSPECTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Therefore, the existing national standard method has the disadvantages of long detection cycle, cumbersome detection steps, high requirements for the experience of the inspectors, and easily leading to missed detection.

Method used

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  • Fluorescent polymerase chain reaction (PCR) primer, probe and method for detecting A, C and G group of hemolytic streptococcus
  • Fluorescent polymerase chain reaction (PCR) primer, probe and method for detecting A, C and G group of hemolytic streptococcus
  • Fluorescent polymerase chain reaction (PCR) primer, probe and method for detecting A, C and G group of hemolytic streptococcus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Design and synthesis of primers and probes.

[0034] The streptococci with beta hemolytic characteristics are mainly group A, B, C and G streptococci, while the streptococci with streptokinase-positive characteristics are group A, C and G streptococci, and the streptococci with bacitracin-positive characteristics are No related reports were found in the literature for which groups, so bacitracin sensitivity experiments were carried out.

[0035] The results are shown in Table 1. If the diameter of the bacitracin disk inhibition zone is greater than 10 mm, it is judged as bacitracin-sensitive, that is, the bacitracin-sensitive test is positive, and is indicated by +; if the bacitracin disk inhibition zone is less than or equal to 10 mm, it is judged as bacitracin-resistant The bacitracin sensitivity test was negative, indicated by -. The results showed that the strains with positive bacitracin test and B hemolytic characteristics were group A, C and G hemoly...

Embodiment 2

[0045] Example 2 Fluorescent PCR detection method.

[0046] Aseptic procedures were performed in a biological safety cabinet.

[0047] Sample processing: Sample dilution and enrichment were performed in a P2 clean room according to the requirements of GB / T 4789.11-2003, and the sample enrichment solution was placed in an incubator at 37°C for 18 hours.

[0048] DNA template: Take 1mL of enrichment solution in a 1.5mL centrifuge tube, centrifuge at 12000rpm for 10min, remove the supernatant, and collect the bacteria. Add 100 μL of sterilized ultrapure water, mix well, and bathe in boiling water at 100°C for 10 minutes. Centrifuge at 12000rpm for 10min, and take the supernatant as the reaction template.

[0049] Reaction system: forward primer (20 μmol / L), 0.4 μL; reverse primer (20 μmol / L), 0.4 μL; probe (20 μmol / L), 0.2 μL; Premix Ex Taq (2×), 10 μL; DNA template, 5uL; add sterilized ultrapure water to make up the reaction system to 20 μL. The 5' end of the probe is lab...

Embodiment 3

[0052] Example 3 Specificity of detection of hemolytic streptococcus by fluorescent PCR.

[0053] According to the fluorescent PCR primers, probes and methods provided by the present invention, 35 common foodborne pathogenic bacteria (Table 2) were amplified to analyze the specificity of the fluorescent PCR detection method. Streptococcus pyogenes (No. 1), Streptococcus equi subsp. zooepidemicus (No. 4), Streptococcus dysgalactiae subsp. equisimilis (No. 5) and Streptococcus equine epidemics (No. 6) according to GB / T 4789.11-2003 Confirmed as hemolytic streptococci. Streptococcus pyogenes (No. 1), Streptococcus equi subsp. zooepidemicus (No. 4), Streptococcus dysgalactiae subsp. Streptococcus (No. 2), Streptococcus uberis (No. 3) and Streptococcus bovis group D (No. 7) were used as negative controls, and sterilized ultrapure water was used as a blank control. Two parallels were set up for each test group.

[0054] CMCC is the abbreviation of China Medical Microbiology Cult...

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Abstract

The invention relates to a fluorescent polymerase chain reaction (PCR) primer, a probe and a method for detecting the whole group of hemolytic streptococcus, belonging to the field of inspection and quarantine. In the primer, hemolysin S of A, C and G group hemolytic streptococcus is used as a target gene site to design a fluorescent PCR primer and a probe and establish a fluorescent PCR detection method for the whole group of the hemolytic streptococcus. The fluorescent PCR primer, the probe and the method are mainly used for detecting the hemolytic streptococcus.

Description

technical field [0001] The invention belongs to the field of inspection and quarantine, in particular to fluorescent PCR primers, probes and methods for detecting whole groups of hemolytic streptococci. Background technique [0002] Hemolytic streptococcus is an important food-borne pathogen, which can cause symptoms such as suppuration and sepsis. The hemolytic streptococcus in the daily inspection is a routine inspection item, and the detection task is heavy. Most of the food safety incidents caused by hemolytic streptococcus are caused by group A hemolytic streptococcus infection, but in recent years, the cases of infection caused by group C and G hemolytic streptococci have shown an increasing trend. [0003] At present, the detection method of hemolytic streptococcus is mainly based on the national standard "GB / T 4789.11-2003 Microbiological Examination of Food Hygiene - Hemolytic Streptococcus Inspection", hereinafter referred to as the national standard method. Acco...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/14C12N15/11G01N21/64
Inventor 林霖杨国武赖心田兰全学严琼英祝仁发李意陈血建陈国培
Owner SHENZHEN ACAD OF METROLOGY & QUALITY INSPECTION
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