Fluorescence quantitative PCR (polymerase chain reaction) detection kit for salmonella and detection method thereof

A detection kit and Salmonella technology, applied in the field of microbial detection, can solve the problems of cumbersome procedures and time-consuming, and achieve high detection sensitivity, low detection false positives, and high specificity

Active Publication Date: 2012-05-09
广州华银医学检验中心有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, many countries, including my country, still use the traditional culture method for the clinical detection of Salmonella. This method is cumbersome and takes a lot of time. It takes about 4-7 days to report the test results, which is far from satisfying the modern rapid detection. need for diagnosis

Method used

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  • Fluorescence quantitative PCR (polymerase chain reaction) detection kit for salmonella and detection method thereof
  • Fluorescence quantitative PCR (polymerase chain reaction) detection kit for salmonella and detection method thereof
  • Fluorescence quantitative PCR (polymerase chain reaction) detection kit for salmonella and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Establishment of Fluorescence Quantitative PCR Detection Method

[0031] 1. Acquisition of specific primers and fluorescent probes:

[0032] Find the fimY gene sequences of various genus Salmonella from GenBank, and perform homology comparison analysis to find the most conserved region of the gene. Using the sequence with the accession number M90677 as a template, specific primers and fluorescent probes based on the Salmonella fimY gene were designed using Primer express 2.0 software. The BLAST analysis of the designed specific primers and probe sequences showed that the set of primers and probes had high specificity. The fluorescent reporter group labeled at the 5' end of the probe is FAM, and the fluorescent quencher group labeled at the 3' end is BHQ-1, which was synthesized by Shanghai Yingjun Biotechnology Co., Ltd.

[0033] The sequence is as follows: (F indicates the upstream primer, R indicates the downstream primer, P indicates the probe)

[0034] F: 5'-TGCG...

Embodiment 2

[0046] Performance Evaluation of Real-time Quantitative PCR Detection Method

[0047] 1. The specificity evaluation of the detection method, the steps are as follows:

[0048](1) Design specific primers and fluorescent probes according to the method described in Example 1, and prepare positive standards and standard gradients.

[0049] (2) Genomic DNA extraction of standard strains: Standard strains (all purchased from Guangdong Microbial Culture Collection Center) Escherichia coli (CMCC44103), Listeria monocytogenes (ATCC19115), Bifidobacterium bifidum (ATCC29521), dysentery Genomic DNA of Shigella (CMCC51252) and Salmonella Enteritidis (ATCC14028) were extracted using a bacterial genome extraction kit (Ningbo Ji Nei Biotechnology Co., Ltd.), and the extraction was performed in strict accordance with the kit requirements.

[0050] (3) Fluorescent quantitative PCR detection was carried out using the genomic DNA of the above-mentioned standard strains as a template, and a posi...

Embodiment 3

[0057] Real-time quantitative PCR detection of Salmonella in clinical samples

[0058] 1. Clinical samples: feces of 100 children with bacterial diarrhea.

[0059] 2. The traditional culture method and the kit established by the present invention are used to detect the above samples respectively, so as to compare the detection results.

[0060] 3. The traditional culture test method is carried out in strict accordance with the requirements of GB / T 4789.4-2003, and the test results are shown in Table 2 below.

[0061] 4. The steps of the detection method established by the present invention are as follows:

[0062] (1) Design specific primers and fluorescent probes according to the method described in Example 1, and prepare positive standards and standard gradients.

[0063] (2) Bacterial genomic DNA extraction from feces samples: The fecal genomic DNA rapid extraction kit (formerly Pinghao (Tianjin) Biotechnology Co., Ltd.) was used to extract in strict accordance with the k...

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Abstract

The invention relates to a fluorescence quantitative PCR (polymerase chain reaction) detection kit for salmonella, comprising a salmonella PCR reaction liquid, a Taq enzyme, a positive control and a negative control. The sequences of the specific primers of the fluorescence quantitative PCR detection kit for salmonella are as follows: the upstream primer is 5'-TGCGCCTGCCGTTATCAC-3'; the downstream primer is 5'-ATTTGAGCTGTTGGTTCAGTAACTC-3'; and the fluorescence probe sequence is 5'-CCAACGCCGCAGATATTTGATCGCC-3'. The detection for salmonella in a clinical excrement by using the kit disclosed by the invention has great specificity, sensitivity and stability, the whole detection process can be finished in 2-3 hours, and salmonella in foods or other cultures also can be rapidly and accurately detected.

Description

technical field [0001] The invention belongs to the field of microbial detection, and in particular relates to a kit for detecting Salmonella developed by utilizing the highly conserved sequence of Salmonella to design specific primers and probes and using fluorescent quantitative PCR technology and a detection method thereof. Background technique [0002] Salmonella is a large genus of a group of Enterobacteriaceae with similar morphology and cultural characteristics. It has various types and is widely distributed. It is one of the common and important zoonotic pathogens, and it is also the most common pathogen that causes food poisoning in humans. . According to statistics, among bacterial food poisoning in various countries in the world, food poisoning caused by Salmonella often ranks first. In my country, food poisoning caused by Salmonella also ranks first in bacterial food poisoning. In order to find the pathogenic bacteria that cause food poisoning, patients’ excreme...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04G01N21/64
CPCY02A50/30
Inventor 温韵洁王晓丹白培胜
Owner 广州华银医学检验中心有限公司
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