DIAGNOSTIC APPLICATION OF POLYMERASE CHAIN-REACTION FOR DETECTION OF EHRLICHIA-RISTICII IN EQUINE MONOCYTIC EHRLICHIOSIS (POTOMAC HORSE FEVER)

被引:19
作者
BISWAS, B
MUKHERJEE, D
MATTINGLYNAPIER, BL
DUTTA, SK
机构
[1] UNIV MARYLAND,VIRGINIA MARYLAND REG COLL VET MED,COLLEGE PK,MD 20742
[2] UNIV MARYLAND,DEPT CHEM & NUCL ENGN,COLLEGE PK,MD 20742
关键词
D O I
10.1128/JCM.29.10.2228-2233.1991
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
Genomic amplification by the polymerase chain reaction (PCR) was used to identify a unique genomic sequence of Ehrlichia risticii directly in DNA isolated from peripheral-blood buffy coat cells of E. risticii-infected horses (Potomac horse fever) and from infected cell cultures. A specific primer pair, selected from a cloned, species-specific, 1-kb DNA fragment of the E. risticii genome as a template, was used for the amplification of the target DNA of 247 bp. The optimal number of 40 PCR cycles, determined by analyzing an amplification profile obtained with a constant Taq polymerase concentration, was used to achieve maximum amplification of the E. risticii DNA segment. Efficient amplification of target DNA was achieved with specimens processed by either the phenol extraction or rapid lysis method. The specificity of the amplified DNA product was confirmed by the proper size (247 bp) and appropriate restriction enzyme cleavage pattern of the amplified target DNA, as well as by the specific hybridization signal obtained by using a PCR-amplified 185-bp internal DNA probe. A 10(5)- to 10(6)-fold amplification of target DNA, which allowed detection of E. risticii from as few as two to three infected cells in culture and from a very small volume of buffy coat cells from infected horses, was achieved. This PCR amplification procedure was found to be highly specific and sensitive for the detection of E. risticii for the study of Potomac horse fever.
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页码:2228 / 2233
页数:6
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