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A high-throughput visual chip for rapid detection of Rickettsia and its application

A high-throughput technology for rickettsia, applied in the field of molecular biology, can solve the problems of long detection cycle, high cost, and inability to quickly detect samples, so as to improve detection coverage, avoid non-specific hybridization, and avoid non-specificity The effect of cross-reactivity

Active Publication Date: 2022-01-25
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Fournier used the discrepancy between the coding gene sequence of Rickettsia OmpA and the known sequence in GenBank to identify the half-point heat group Rickettsia, and then compared the five different genes of rrs, gltA, ompA, ompB and sca4 at the same time. Gene, comprehensive comparison results for typing, it was found that MLST has greatly improved the sensitivity of Rickettsia detection and the reliability of typing, but the detection cycle is long, the cost is high, and it is impossible to quickly detect a large number of samples at the same time

Method used

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  • A high-throughput visual chip for rapid detection of Rickettsia and its application
  • A high-throughput visual chip for rapid detection of Rickettsia and its application
  • A high-throughput visual chip for rapid detection of Rickettsia and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Preparation of Rickettsia species-specific microarray gene chip

[0049] 1. Materials

[0050] Rickettsia probes and primers: Synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

[0051] Punctual nylon membrane: (Nylon Membranes, positively charged, Roche Company): purchased from Sigma-Aldrich Company.

[0052] Precipitation-type TMB chromogenic solution: purchased from Beijing Aobolong Immunotechnology Co., Ltd.

[0053] Hybridization solution: purchased from Suzhou Institute of Nanotechnology, Chinese Academy of Sciences.

[0054] Horseradish peroxidase-labeled streptavidin (streptavidin-HRP): purchased from Thermo Fisher Scientific;

[0055] Blood and tissue sample DNA extraction kit (DNeasy 96 Blood & Tissue Kit): purchased from Qiagen, Germany;

[0056] Hybridization stock solution M1, M2 and M3: purchased from Shanghai Jieyuan Biotechnology Co., Ltd. Prepare chip working solutions according to the ratio instructions (see above): hybridization ...

Embodiment 2

[0078] The construction of embodiment 2 rickettsia positive plasmid standard product

[0079] 1. Materials:

[0080] (1) The 10 standard positive rickettsias constructed include:

[0081] Rickettsia Prowazeki, Rickettsia mooseri, Rickettsia Conorii, Rickettsia Heilongjiangii, Rickettsia ( Rickettsia Rickettsii, Rickettsia Sibirica, Ehrlichia Chaffeensis, Ehrlichia Canis, Orientia tsutsugamushi, and Coxiella bainii (Coxiella Burnetii).

[0082] (2) pMD18-T Vector kit, purchased from TAKARA, item number: D101A

[0083] (3) Gel extraction kit: Gel Extraction Kit, purchased from OMEGA, article number: D2500.

[0084] 2. Preparation of Rickettsia positive plasmid standard:

[0085] (1) The PCR target sequence was amplified with specific multiple primers for the Rickettsia 16S rRNA gene, and the PCR products were detected by agarose gel electrophoresis. The agarose gel recovery kit for the target band was recovered and purified, and the purified PCR product was submitted to Sa...

Embodiment 3

[0092] Example 3 Specific detection of Rickettsia high-throughput visualized gene chip

[0093] 1. Materials:

[0094] Plasmid extraction kit: OMEGAbio-tek E.Z.N.A. Plasmid DNA Mini Kit (Cat. No.: D6943-01).

[0095] Rickettsia primers: commissioned by Sangon Bioengineering (Shanghai) Co., Ltd. to synthesize.

[0096] The Rickettsia genus-specific chip prepared in this experiment; the Rickettsia species-specific chip.

[0097] Precipitation-type TMB chromogenic solution: purchased from Beijing Aobolong Immunotechnology Co., Ltd.

[0098] Horseradish peroxidase-labeled streptavidin (streptavidin-HRP): purchased from Thermo Fisher Scientific.

[0099] Hybridization stock solutions M1, M2 and M3: purchased from Shanghai Jieyuan Biotechnology Co., Ltd. Prepare chip working solutions according to the ratio instructions (see above): hybridization solution A, washing solution B and washing solution C.

[0100] Horseradish peroxidase-labeled streptavidin: Abcam, UK.

[0101] Ricke...

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Abstract

The invention relates to the field of molecular biology, and specifically discloses a chip for high-throughput visual rapid detection of rickettsia and its application. The invention aims at realizing high-throughput species- and genus-specific detection by designing rickettsia genus-specific and species-specific probes. And by artificially synthesizing biotin-labeled primers and probes, using streppenicillin-horseradish peroxidase to combine with labeled biotin, and further performing color reaction with precipitated TMB to obtain visual detection results. The present invention can be used for the rapid diagnosis of following 10 kinds of rickettsias: Rickettsia praustii, Rickettsia moschii, Rickettsia consoni, Rickettsia heilongjiang, Rickettsia rickettsia, Siberia Rickettsia, Ehrlichia chaffeensis, Ehrlichia canis, Orientia tsutsugamushi, and Coxiella beinii.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a chip for high-throughput visual rapid detection of rickettsia and its application. Background technique [0002] Rickettsiae (rickettsiae) is a kind of obligate intracellular parasitic Gram-negative bacteria except for a very small number, and the rickettsiae caused by it is an important class of zoonotic natural foci diseases. It is sporadic and seasonal worldwide. Many arthropods such as lice, fleas, ticks, mites, etc. can be used as the transmission medium of Rickettsia infection. With the development of international tourism, rickettsial disease is considered to be one of the important infectious diseases that threaten the health of tourists. In recent years, the continuous emergence of new species of spotted fever and the prevalence of new Rickettsia such as Ehrlichia remind us of the possibility of large-scale epidemics and sudden outbreaks of rickettsial di...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/04C12N15/11C40B40/06C12R1/01
CPCC12Q1/689C40B40/06Y02A50/30
Inventor 何诚王艺晖郭永霞沈志强曲光刚
Owner CHINA AGRI UNIV
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