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
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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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