RPA detection method, special primers and probe for detecting Rickettsia rickettsii, and uses of RPA detection method in detection of Rickettsia rickettsii

A Rickettsia rickettsii and probe technology, applied in the biological field, can solve the problems of limited practical application range, time-consuming samples, complex processing, etc., and achieve the effects of shortening detection time, saving detection time, and broad application prospects.

Active Publication Date: 2018-02-23
李佳萌
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these methods have disadvantages such as high cost, special equipment, time-cons...

Method used

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  • RPA detection method, special primers and probe for detecting Rickettsia rickettsii, and uses of RPA detection method in detection of Rickettsia rickettsii
  • RPA detection method, special primers and probe for detecting Rickettsia rickettsii, and uses of RPA detection method in detection of Rickettsia rickettsii
  • RPA detection method, special primers and probe for detecting Rickettsia rickettsii, and uses of RPA detection method in detection of Rickettsia rickettsii

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1, design and screening of Rickettsia rickettsia primers and probes

[0040] (1) Design of primers and probes

[0041] The inventor analyzed and determined that the specific sequence of Rickettsia rickettsii used in the present invention is the target gene through file retrieval. The known template gene sequence was obtained from the NCBI database, that is, the nucleotide sequence shown in SEQ ID NO.: 1, and the above sequence was synthesized by Nanjing KingScript Biotechnology Co., Ltd., as a positive plasmid, in the subsequent primers It is used as a template in the process of probe screening and optimization of reaction system. According to the RPA primer and probe design principles, Prime 5 software was used to design 3 sets of upstream primers, 5 sets of downstream primers and 1 probe, as shown in Table 1.

[0042] Table 1 Primers and probes

[0043] Primers / Probes

Sequence (5'→3')

rrf53

CTAGCAATAATCTGTGTTATTTGATAAAAT

rrf...

Embodiment 2

[0054] Embodiment 2: Optimization of RPA reaction system, amplification and detection conditions

[0055] In the process of primer screening, there are still false positives in the detection of lateral flow nucleic acid detection test strips, so the RPA reaction system, amplification and detection conditions need to be optimized

[0056] (1) Primer probe concentration

[0057] Set the concentration of the reverse primer to 10 μmol / L, the concentration gradient of the probe to 10 μmol / L, 5 μmol / L, and 2.5 μmol / L, and combine one concentration of the reverse primer with three concentrations of the probe, Three groups of combinations were combined, and a set of negative controls was set for each combination. RPA amplification was carried out at 37°C, and after the amplification was completed, the products were detected with lateral flow nucleic acid detection strips, and the combination with the best amplification effect and no false positives was screened out.

[0058] Table 3...

Embodiment 3

[0065] Embodiment 3: Sensitivity evaluation of RPA detection

[0066] Dilute the positive plasmid to 10 4 - 10 0 A series of different concentrations such as 1 / μL, 1 μL each was added to the reaction system determined in Example 2, and the primer combination screened out was used to perform RPA on the above-mentioned templates with different copy numbers using the amplification and detection conditions determined in Example 2. Detection, observe the sensitivity of RPA detection.

[0067] As a result, the above samples were all positive from the copy number of 10 copies / μL, indicating that the sensitivity of the RPA detection method of the present invention reaches 10 copies / μL.

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Abstract

The invention provides an RPA detection method, special primers and a probe for detecting Rickettsia rickettsii, and uses of the RPA detection method in detection of Rickettsia rickettsii. According to the present invention, the special primers and the probe are designed based on the conserved sequence of a Rickettsia rickettsii gene, and have the oligonucleotide sequences represented by SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4; the novel constant temperature amplification technology RPA is firstly applied in the detection of Rickettsia rickettsii, wherein the enzymatic reaction process of DNAreplication in vivo is simulated, the DNA template is amplified by using the specific enzyme and protein combination including recombinase, single-stranded binding protein and DNA polymerase so as toamplify the specific nucleic acid sequence at the constant temperature of 37 DEG C, and the amplification product can be subjected to visualized discrimination through the lateral chromatography nucleic acid detection test paper strip; and the established detection method has the sensitivity of 6 copies/[mu]L, has high specificity, has low requirement on hardware equipment, can complete the detection within 30 min, does not require the complicated sample treatment, is suitable for on-site detection, and is suitable for popularization and application.

Description

technical field [0001] The invention belongs to the field of biotechnology, relates to the molecular biology of Rickettsia rickettsia, relates to a method for detecting Rickettsia rickettsia and its application, and particularly relates to a technique of using recombinase polymerase constant temperature amplification (RPA Technology) rapid detection method for Rickettsia rickettsiae, its special primers and probes and its application. Background technique [0002] Rickettsia rickettsiae ( Rickettsia rickettsii , Rr), commonly known as spotted fever, is the pathogen of Rocky Mountain spotted fever. It is a small obligate intracellular gram-negative bacterium that has strong resistance to the external environment and a long survival time. Humans and animals are generally susceptible, and can pass through Aerosols are widespread and are among the most lethal, biologically agentized pathogens. Rickettsia rickettsii mainly invades arteriovenous endothelial cells, causing vascul...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/04C12N15/11C12R1/01
CPCC12Q1/6844C12Q1/689C12Q2521/507C12Q2522/101C12Q2525/186
Inventor 齐永李越希邵银秀尹琼曹勇平陈红霞陈乐如饶继先李佳萌沈万鹏李素芹徐亭亭杨彬彬
Owner 李佳萌
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