RESUMO
A rapid and sensitive multiplex PCR has been developed for the diagnosis of multiple parasitic infection in human blood. Infection is detected by a single multiplex PCR reaction containing two pairs of oligonucleotide primers whereby each primer is specific for each parasite species. These primer sets amplified 400 and 450-bp fragments for Wuchereria bancrofti and 208-bp fragment for Plasmodium falciparum. The PCR products derived from each parasite species were visualized in ethidium bromide-stained agarose gels, therefore allowing the rapid identification of any, or all, of the two human parasites, if present, in a single amplification reaction. This multiplex PCR was very sensitive with the ability to detect the presence of as little as 10 pg of parasite DNA. The primers used in this multiplex PCR also showed highly specific amplification of each respective parasite DNA without the presence of non-specific and non-target PCR products. This multiplex PCR system was used to analyse 36 human blood samples of Myanmar workers in the endemic area at Tak Province, Thailand. Two samples showed the multiple infection, 27 samples were either infected with W. bancrofti or P. falciparum and seven samples were negative for both methods. The high sensitivity, specificity and rapidity of this multiplex PCR method make it suitable for large-scale epidemiological studies and following of drug treatment.
Assuntos
DNA de Protozoário/sangue , Plasmodium falciparum/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Wuchereria bancrofti/isolamento & purificação , Adolescente , Adulto , Animais , Primers do DNA , DNA de Protozoário/genética , Humanos , Plasmodium falciparum/genética , Sensibilidade e Especificidade , Tailândia , Wuchereria bancrofti/genéticaRESUMO
Twenty-eight field isolated Theileria parasite DNAs obtained from dairy and beef cattle in distinct geographical areas of Thailand were characterized by using polymerase chain reaction (PCR) amplification with six sets of oligonucleotide primers. Three sets of them were modified from two genes of immunodominant major piroplasm surface protein (MPSP) coding for 32 kDa (p32) of T. sergenti and 33/34 kDa (p33/34) of T. buffeli, and MPSP of Theileria spp.(Thai-isolate). The other three sets of primers were basically generated from three alleles of MPSP which were specific for Japanese T. sergenti-Ikeda stock (I-type), Japanese T. sergenti-Chitose stock (C-type) and Australian T. buffeli-Warwick stock (B1-type), respectively. The results indicated that 14 out of 28 isolates were amplified by the Thai-specific primer whereas 6 isolates were amplified by the p32 specific primer and the other 5 isolates were amplified by the p32 and Thai-specific primers. In addition, by using the allele-specific PCR, 14 out of 28 isolates contained C-type MPSP whereas 3 isolates contained B1 type parasites. Interestingly, 20 out of 28 isolates could be amplified by the Thai-specific primer. The majority of Theileria parasites distributed in Thailand contained Thai type parasites, whereas C-type parasites showed the mixed population with B1 and Thai type parasites. No I type parasite was detected.
Assuntos
Variação Genética/genética , Proteínas de Protozoários/genética , Theileria/genética , Theileriose/parasitologia , Alelos , Animais , Bovinos , Primers do DNA/química , DNA de Protozoário/química , Eletroforese em Gel de Ágar , Feminino , Proteínas de Membrana/química , Proteínas de Membrana/genética , Peso Molecular , Reação em Cadeia da Polimerase/veterinária , Proteínas de Protozoários/química , Tailândia , Theileria/química , Theileria/classificaçãoRESUMO
A method was developed to obtain reproducible DNA fingerprints from five distinct purified benign Theileria genomic DNAs by PCR-based amplification. Randomly amplified polymorphic DNA (RAPD) profiles were obtained from 10 randomly designed 12-mers. However, nine of the 10 primers could generate the difference in RAPD-PCR profiles which allowed discrimination of Theileria species. The method has advantage of being simple, fast and sensitive for diagnosis and characterization of the parasites since it does not require prior DNA sequence information to construct species-specific probes or primers. The results are also beneficial for a proper understanding of the epidemiology and designing rational control programmes for Theileriosis in Asian and South-East Asian countries.
Assuntos
DNA de Protozoário/análise , Theileria/classificação , Theileriose/parasitologia , Animais , Sudeste Asiático/epidemiologia , Austrália/epidemiologia , Bovinos , Impressões Digitais de DNA/métodos , Impressões Digitais de DNA/veterinária , Primers do DNA/química , Eletroforese em Gel de Ágar/veterinária , Genoma de Protozoário , Japão/epidemiologia , Técnica de Amplificação ao Acaso de DNA Polimórfico/veterinária , Reprodutibilidade dos Testes , Especificidade da Espécie , Theileria/genética , Theileriose/epidemiologiaRESUMO
A method was developed to obtain reproducible DNA fingerprints from isolates of Trypanosoma evansi by PCR-based amplification using arbitrary primers (AP-PCR). Only one out of 10 randomly designed 12-mer primers generated DNA fingerprint profiles that revealed intra-species differences in T. evansi. The technique was applied in association with parasitological and serological examinations to investigate animal-to-animal transmission during an outbreak of surra in Thailand. The AP-PCR method has the advantage of being simple, fast and sensitive to diagnosis and characterization of the parasites since it does not require prior DNA sequence information. The technique should prove useful for the proper understanding of epidemiology and for designing rational control programs for trypanosomosis.
