Kit and method for detecting anaplasmosis and rickettsioses based on loop-mediated isothermal amplification (LAMP)

A technology for detecting and grouping Rickettsia, which is applied in the field of Rickettsia detection kits and can solve the problems of low sensitivity and specificity

Inactive Publication Date: 2011-11-23
ICDC CHINA CDC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a method for detecting spotted fever group Rickettsia and Anaplasma phagocytophilum with strong specificity and high sensitivity for the existing diagnostic methods that are time-consuming, require special instruments, and have low sensitivity and specificity. High, fast response, easy to operate, and does not require special instrument detection methods and kits, which can be better applied to clinical diagnosis in areas with insufficient conditions and equipment

Method used

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  • Kit and method for detecting anaplasmosis and rickettsioses based on loop-mediated isothermal amplification (LAMP)
  • Kit and method for detecting anaplasmosis and rickettsioses based on loop-mediated isothermal amplification (LAMP)
  • Kit and method for detecting anaplasmosis and rickettsioses based on loop-mediated isothermal amplification (LAMP)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Detection of spotted fever group Rickettsia

[0037] 1. Design of LAMP primers for spotted fever group Rickettsia ompB gene

[0038] Rickettsia sp.BJ-90 (AY331393) sequence was used as a template in the conserved region obtained from the alignment, using the LAMP primer design software (Primer Explorer software, version 4.0) designed LAMP primers in this region, and designed 3 sets of primers:

[0039] Table 1 LAMP-specific primers for spotted fever group Rickettsia ompB gene

[0040]

[0041]

[0042] The designed primers were sent to Shanghai Sangong for synthesis and purified by HPLC.

[0043] 2. Standard plasmid preparation for Rickettsia spotted fever group

[0044] According to the region where the 3 sets of LAMP primers are located, use the sequence of Rickettsia sp.BJ-90 (GenBank: AY331393) as a template, use Primer Premier 5 software to design 2 sets of primers, and make standard plasmids: OPBC1 (5'-ATAATGGCGCAGGTAGAGC-3') and OPBC2 (5'-CAC...

Embodiment 2

[0070] Example 2 Detection of Anaplasma phagocytophilum

[0071] 1. Design of LAMP primers for Anaplasma phagocytophilum msp2 gene

[0072] The Anaplasma msp2 gene sequence was retrieved from Genbank and compared using DNAstar software. In the conserved region obtained by the comparison, the A. phagocytophilum isolate EQ6 D22 (AY763471) sequence was used as a template, and the LAMP primer design software (Primer Explorer software, version 4.0) designed LAMP primers in this region, and designed 3 sets of primers:

[0073] Table 5 Design of LAMP primers for Anaplasma phagocytophilum msp2 gene

[0074]

[0075]

[0076] The designed primers were sent to Shanghai Sangong for synthesis and purified by HPLC.

[0077] 2. Preparation of standard plasmids for the detection gene of Anaplasma phagocytophilum msp2

[0078] According to the region where the 3 sets of LAMP primers are located, use the sequence of A.phagocytophilum isolate EQ6 D22 (AY763471) as a template, use Prime...

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Abstract

The invention discloses a kit and method for detecting anaplasmosis and rickettsioses based on LAMP. The invention relates to a molecular biological detection technology for anaplasmosis and rickettsioses, provides specific primers which are designed to be directed to ompB gene of spotted fever group rickettsia (SFGR) and msp2 gene of anaplasma phagocytophilum, and provides a detection kit for SFGR and anaplasma phagocytophilum by using LAMP. The kit has the characteristics of rapidness, sensitivity, high specificity, no experimental condition restrictions, etc., and is applicable to clinicaldetection for spotted fever and granulocytic anaplasmosis, especially in rural areas where medical conditions are limited. The invention also discloses a method for detecting rickettsioses with the kit.

Description

technical field [0001] The invention relates to a detection kit and a method for Rickettsia. Specifically, the present invention relates to a kit for detecting pathogenic microorganisms using loop-mediated isothermal amplification technology (Loop-mediated isothermal amplification, LAMP), and a detection method thereof. Background technique [0002] Loop-mediated isothermal amplification (LAMP) is a new nucleic acid amplification method. The LAMP method has the characteristics of high sensitivity, high specificity and easy operation, and can amplify the target gene by 10% within one hour under constant temperature conditions. 9 times. Spotted fever is a tick-borne rickettsial disease caused by a type of obligate intracellular parasitic spotted fever group rickettsiae (SFGR) [P.Parola, C.D.Paddock, D.Raoult, Tick-borne rickettsioses around the world : emerging diseases challenging old concepts, Clin Microbiol Rev, vol.18, no.4, pp.719-756, 2005.]. The disease is widely di...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12R1/01
Inventor 张丽娟潘磊
Owner ICDC CHINA CDC
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