Multiplex polymerase chain reaction based assay for the detection of Babesia bigemina, Babesia bovis and Anaplasma marginale DNA in bovine blood
| Autor: | J V Figueroa, L.P. Chieves, G S Johnson, G. M. Buening |
|---|---|
| Rok vydání: | 1993 |
| Předmět: |
DNA
Bacterial Male Anaplasmosis Anaplasma Molecular Sequence Data Babesia Cattle Diseases Polymerase Chain Reaction law.invention Nucleic acid thermodynamics law Babesiosis Multiplex polymerase chain reaction Animals Babesia bigemina Polymerase chain reaction DNA Primers Southern blot Base Sequence General Veterinary biology Babesia bovis General Medicine DNA Protozoan biology.organism_classification Virology Molecular biology Agarose gel electrophoresis Cattle Parasitology |
| Zdroj: | Veterinary Parasitology. 50:69-81 |
| ISSN: | 0304-4017 |
| DOI: | 10.1016/0304-4017(93)90008-b |
| Popis: | A highly sensitive polymerase chain reaction (PCR) based method was developed to detect, in the same blood sample, DNA of hemoparasites frequently found together infecting cattle in tropical and subtropical areas. Bovine blood containing equal parasitemias of Babesia bigemina, B. bovis and Anaplasma ma marginale infected erythrocytes was mixed to standardize the test. Twenty microliters of 10-fold dilutions from the pooled blood sample were resuspended in PCR mixture buffer containing each of the species-specific sets of primers. Group I primers (BiIA/IB, BoF/R and Am9.10) which specifically bind B. bigemina, B. bovis and A. marginale DNA were used to amplify a fragment of DNA from genomic parasite DNA. Group II nested primers (BiIAN/IBN, BoFN/RN and Am11/12) were used to prepare, via incorporation of digoxigenin-11-dUTP by PCR, nonradioactive probes specific for internal sequences present in DNA amplified with Group I primers. Agarose gel electrophoresis and Southern blot hybridization studies showed that by using Group I primers, DNA fragments of 278 bp, 350 bp and 200 bp were specifically amplified in samples containing B. bigemina, B. bovis and A. marginale DNA, respectively. The analytical sensitivity of the multiple PCR test, as evaluated by nucleic acid hybridization with the nonradioactive probe, was 0.00001%, 0.00001% and 0.00001% infected erythrocytes for B. bigemina, B. bovis and A. marginale, respectively. Blood collected from cattle previously inoculated with B. bovis (4 years), A. marginale (2 years) and B. bigemina (1 year) was demonstrated to be latently infected by using the Multiplex PCR test. |
| Databáze: | OpenAIRE |
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