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Primer probe composition and kit for real-time fluorescence RAA detection of cyclosporidia DNA and application of primer probe composition and kit

A primer probe and real-time fluorescence technology, which is applied in the field of molecular biology detection of parasites, can solve the problems of missed detection and misdiagnosis, high cost, limited timely detection and traceability, etc.

Pending Publication Date: 2021-04-13
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 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the small size of oocysts, direct smear method and microscope examination have a certain degree of missed detection and misdiagnosis; the equipment of flow cytometry is complicated and expensive, and it is not suitable for large-scale application
The above methods have certain limitations in the detection of Cyclospora, which limits the timely detection and traceability of Cyclospora outbreaks

Method used

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  • Primer probe composition and kit for real-time fluorescence RAA detection of cyclosporidia DNA and application of primer probe composition and kit
  • Primer probe composition and kit for real-time fluorescence RAA detection of cyclosporidia DNA and application of primer probe composition and kit
  • Primer probe composition and kit for real-time fluorescence RAA detection of cyclosporidia DNA and application of primer probe composition and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1 screening primer probe group

[0029] The invention provides a primer, a probe and a kit for detecting Cyclospora based on the RAA fluorescence method, and the implementation method has the following steps:

[0030] 1) Primer probe design and selection of fluorescent modifying groups and fluorescent quenching groups on the probes;

[0031] 2) synthetic primer probe;

[0032] 3) Synthetic positive plasmid;

[0033] 4) Prepare positive plasmid standard;

[0034] 5) Primer screening;

[0035] 6) Confirmation of the best primer combination;

[0036] 7) Extract Cyclospora sample DNA for verification;

[0037] According to the name of the sample Cyclospora cayetanensis, the sequence information of multiple Cyclospora cayetanensis 18S ribosomal RNA genes was obtained through the National Center for Biotechnology Information (NCBI), and DNAMAN software was used for homology analysis and Blast sequence analysis to screen out The highly conserved sequence of Cy...

Embodiment 2

[0082] The composition of the kit:

[0083] The design of primers and probes is the same as that of the primers and probes in the specific embodiment, and is not repeated here. The sequences of the primers and probes are the same as those of the primers and probes in the specific embodiment, and are not repeated here To repeat, the modification of the fluorescent reporter group, quencher group and tetrahydrofuran residue of the probe is the same as that in the specific embodiment, and will not be repeated here.

[0084] The composition of the kit is shown in Table 6.

[0085] Table 6 Kit Composition

[0086]

[0087] Transfer 5 μL of the recombinant plasmid to Escherichia coli and extract it at a concentration of 10 10 Copies / μL DNA plasmids are used to make working standards of different gradients, which are:

[0088] Standard work piece 1, containing 1.0×10 5 Copies / µL non-infectious DNA fragment of the 18S rRNA gene of Cyclospora.

[0089] Standard work 2, containin...

Embodiment 3

[0115] The composition of the kit is the same as in Example 2.

[0116] Sample source and DNA extraction:

[0117] The sample was provided by the National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention. It was a stool sample from a patient with clinical diarrhea. DNA was extracted using a commercial stool sample DNA extraction kit. The extraction procedure was carried out according to the kit instructions. Store at ℃ for later use.

[0118] Reaction system preparation: Take 3 reaction tubes and operate as follows respectively, absorb 47 μL of reaction buffer, add 2 μL of primer-probe composition, and mix well; In the reaction tube, fully dissolve the freeze-dried powder and mix well; add 1 μL of negative quality control substance and 1 μL of extracted sample DNA to the three prepared reaction tubes as templates, and mix well for each reaction tube. The total volume of each reaction tube is 50 μL;

[0119] The detection instrument adopts t...

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Abstract

The invention discloses a primer probe composition and probe for real-time fluorescence RAA detection of cyclosporidia DNA. The primer probe composition comprises an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.2. The nucleotide sequence of the probe is shown as SEQ ID NO. 7. In addition, the invention also discloses a kit containing the primer probe composition and application of the primer probe composition. The cyclosporidia can be rapidly and effectively detected, the detection accuracy, the specificity and the sensitivity are high, and the detection kit is sensitive, stable and effective.

Description

technical field [0001] The invention relates to the technical field of molecular biology detection of parasites, in particular to a primer set, kit and method for qualitative detection of Cyclospora DNA. Background technique [0002] Cyclospora (Cyclospora, also known as Cyclospora) is a zoonotic intestinal pathogenic protozoa that can cause severe diarrhea and other gastrointestinal diseases in immunocompetent or immunocompromised individuals. It is also accompanied by symptoms such as loss of appetite, fatigue, abdominal distension, abdominal pain, nausea, vomiting, weight loss, and even death in severe cases. The life cycle of Cyclospora belongs to the typical single-host type, that is, asexual reproduction and sexual reproduction are completed in one host. The Cyclospora oocysts in the feces just excreted by the host are not infective, and the oocysts need to go through a sporulation process at a suitable temperature (22-32°C) for about 1-2 weeks to become infectious. ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6893C12Q1/6844C12N15/11C12R1/90
CPCC12Q1/6893C12Q1/6844C12Q2521/507C12Q2522/101C12Q2563/107
Inventor 沈玉娟姜岩岩许洁曹建平应清界刘燕红顾赛艺
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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