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Multiplex PCR detection kit and detection method for newly-emerged intestinal protozoa

A new protozoa and kit technology, applied in the field of primers for Giardia, simultaneous detection of Cryptosporidium, Cyclospora and Microsporidia, can solve the problem of no simultaneous detection, and achieve far-reaching economic value and social benefits , Save the cost of testing and avoid the high effect of false positives

Active Publication Date: 2015-06-24
中国疾病预防控制中心寄生虫病预防控制所国家热带病研究中心
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Problems solved by technology

There is currently no multiplex PCR method for the simultaneous detection of Cryptosporidium, Microsporidium, Cyclosporidium, and Giardia in stool samples

Method used

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  • Multiplex PCR detection kit and detection method for newly-emerged intestinal protozoa
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  • Multiplex PCR detection kit and detection method for newly-emerged intestinal protozoa

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Embodiment 1

[0069] 1. Materials and methods

[0070] 1. Materials

[0071] 1.1 sample

[0072] 1.1.1 Positive samples of Cryptosporidium, Giardia, Cyclosporidium and Microsporidium were isolated from the laboratory, stored in potassium dichromate, and stored at 4°C.

[0073] 1.1.2 On-site samples were collected from diarrhea patients and animal feces samples in hospitals in Shanghai, Jiangsu and other places.

[0074] 1.2 Reagents

[0075] Fecal DNA extraction kit was purchased from Qiagen, USA; PCR mix was purchased from Bio-Serve; PCR product recovery kit QIAquick Gel Extraction Kit was purchased from Qiagen, Germany; pMD19-T Vector was purchased from Takara; plasmid DNA extraction kit Purchased from Tiangen (Beijing) Biochemical Technology Co., Ltd.

[0076] 2. Method

[0077] 2.1 Primer design

[0078] Multiple gene sequences of four protozoa were downloaded from GenBank for comparison, and primers were designed. See Table 1 for primer names and sequences.

[0079] Table 1: Mul...

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Abstract

The invention discloses a multiplex PCR detection kit for newly-emerged intestinal protozoa, which is used for simultaneously detecting cryptosporidium, giardia intestinalis, cyclospora cayetanensis and microsporidia. The kit comprises the following primers: specific primers for cryptosporidium: shown in SEQ ID NO.1 and SEQ ID NO.2; specific primers for giardia intestinalis: shown in SEQ ID NO.3 and SEQ ID NO.4; specific primers for cyclospora cayetanensis: shown in SEQ ID NO.5 and SEQ ID NO.6; and specific primers for microsporidia: shown in SEQ ID NO.7 and SEQ ID NO.8. In addition, the invention also discloses a multiplex PCR detection method for cryptosporidium, giardia intestinalis, cyclospora cayetanensis and microsporidia. Various validations show that the multiplex PCR detection kit and detection method disclosed by the invention have the characteristics of rapidness, sensitivity, and strong specificity, and solve the cumbersome steps of multiplex PCR detection, and through one-time reaction, an effect of simultaneously detecting multiple genes and determining pathogens can be achieved, so that the workload is greatly reduced, the detection time is shortened, and the detection cost is reduced, therefore, the multiplex PCR detection kit and detection method disclosed by the invention have a greater development trend.

Description

technical field [0001] The invention relates to the technical field of agriculture and animal quarantine, in particular to a detection method for Cryptosporidium, Giardia, Cyclosporidium and Microsporidium, in particular to a detection method for Cryptosporidium, Giardia, Cyclosporidium and Microsporidium Multiplex PCR detection method and kit for sporozoites. Furthermore, the present invention relates to primers for the simultaneous detection of Cryptosporidium, Giardia, Cyclosporidium and Microsporidium. Background technique [0002] Cryptosporidium, Giardia lamblia (referred to as Giardia), Cyclosporidium and Microsporidium are new zoonotic intestinal protozoa, mainly transmitted by water, causing consumptive diarrhea, and are important public health issues facing the world. The health problem is also one of the most difficult problems faced by mankind so far; it can break out and cause serious public health emergencies. Human infection is mainly caused by ingestion of ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/90
CPCC12Q1/686C12Q1/6893C12Q2600/16C12Q2537/143C12Q2565/125
Inventor 沈玉娟曹建平刘华袁忠英姜岩岩尹建海王燕娟
Owner 中国疾病预防控制中心寄生虫病预防控制所国家热带病研究中心
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