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Quadruple fluorescent quantitative polymerase chain reaction (PCR) detection kit for diarrheagenic escherichia coli

A detection kit and fluorescent quantitative technology, applied in the direction of fluorescence/phosphorescence, microbial measurement/inspection, biochemical equipment and methods, etc., can solve the problems of cumbersome operation, long time-consuming, time-consuming and labor-intensive, etc.

Active Publication Date: 2012-04-04
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0003] In view of the increasing importance of diarrhea-causing Escherichia coli in public health, the detection of various diarrheal bacteria in the surveillance of gastroenteritis and food poisoning is not only time-consuming and laborious but also a waste of clinical specimens and reagents
Routine detection is mainly based on traditional culture methods, which are not only cumbersome to operate, time-consuming, but also easy to contaminate

Method used

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  • Quadruple fluorescent quantitative polymerase chain reaction (PCR) detection kit for diarrheagenic escherichia coli
  • Quadruple fluorescent quantitative polymerase chain reaction (PCR) detection kit for diarrheagenic escherichia coli
  • Quadruple fluorescent quantitative polymerase chain reaction (PCR) detection kit for diarrheagenic escherichia coli

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Embodiment 1

[0045] 1 Materials and methods

[0046] 1.1 Strains and clinical specimens:

[0047] Diarrhea-causing Escherichia coli O157:H7 and O104:H4 bacteria (the strains were identified by automatic biochemical analyzer and agglutinated by Japanese Shengken and Danish serum) were purchased from the Chinese Center for Disease Control and Prevention. Clinical samples were obtained from fecal specimens of recent patients. After sample collection, they were stored at low temperature and transported to the laboratory in time.

[0048] 1.2 Primers and probes

[0049] The gene sequences of diarrhea-causing Escherichia coli O157:H7 and O104:H4 from around the world were downloaded from the NCBI gene bank in the United States. The homology comparison was carried out, and specific primers and Taqman probes were designed in the conserved gene region of the corresponding genome. The sequences are as follows:

[0050] O157 upstream primer-F: 5'-TGGCATGACGTTATAGGCTACAAT-3'

[0051] O157 Downstre...

Embodiment 2

[0079] Example 2 Detection of clinical samples

[0080]Using the Diarrhea Syndrome Questionnaire of the "Eleventh Five-Year" Major Project-Infectious Disease Pathogen Monitoring Technology Platform, 800 outpatient diarrhea specimens (daily Defecation 3 or more times, and the stool is loose stool, watery stool, mucus stool or pus and blood stool and other characteristics have changed) for testing. Bacterial DNA is directly extracted from the collected diarrhea clinical samples, and the fluorescent PCR method of the present invention is used to detect the target pathogenic bacteria; at the same time, the conventional culture method is used to conduct parallel experiments. Results The fluorescent PCR method detected one part of O157:H7 and two parts of O104:H4. The fluorescent PCR method of the present invention has a higher positive rate than the conventional culture method and is easy to operate. The verification of the fluorescence PCR method of the present invention for clin...

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Abstract

The invention provides a quadruple fluorescent quantitative polymerase chain reaction (PCR) detection kit for diarrheagenic Escherichia coli. The quadruple fluorescent quantitative PCR detection kit consists of fluorescent quantitative PCR reaction liquid, a standard substance O157, a standard substance H7, a standard substance O104, a standard substance H4 and a reference substance, wherein the fluorescent quantitative PCR reaction liquid comprises PCR reaction buffer solution (which contains a mixture of magnesium chloride and deoxyribose nucleotide triphosphate, heat-resisting thermus aquaticus deoxyribonucleic acid (Taq DNA) polymerase and the like), special upstream and downstream primers and a probe. In the quadruple fluorescent quantitative PCR detection kit, O157:H7 and O 104:H4 type bacteria of the diarrheagenic Escherichia coli can be detected from a sample; and compared with the conventional ordinary PCR method and single fluorescent quantitative PCR method, the quadruple fluorescent quantitative PCR detection kit has the advantages of simplicity, convenience, quickness and accuracy; and the detected bacteria are quantitated accurately in real time, and the diagnosis of suspected patients infected with the bacteria clinically is clarified early, so that the treatment scheme is formulated in time to lower the death rate.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a nucleic acid detection method for simultaneous detection of diarrhea-causing Escherichia coli O157:H7 and O104:H4 serotypes in fecal specimens of patients with gastroenteritis and diarrhea by quadruple real-time fluorescence quantitative PCR in the same reaction tube , which can be applied to laboratory emergency detection of outbreaks caused by diarrhea-causing Escherichia coli. Background technique [0002] Diarrhea-causing Escherichia coli is called diarrhea-causing Escherichia coli. According to virulence factors, pathogenic mechanisms and epidemiological characteristics, diarrhea-causing Escherichia coli is usually divided into 5 categories, namely enteropathogenic Escherichia coli (EPEC) , Enterotoxigenic Escherichia coli (ETEC), Enteroinvasive Escherichia coli (EIEC), Enteroaggregative Escherichia coli (EAEC), and Enterohemorrhagic Escherichia coli (EHEC). Following EHEC infe...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/10C12Q1/06G01N21/64
Inventor 陈瑜陈晓郑书发孔海深
Owner ZHEJIANG UNIV
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