A rapid polymerase chain reaction based method for identification of the Anopheles dirus sibling species.

A simple polymerase chain reaction (PCR) based method was developed to differentiate the Anopheles dirus, species A, B, C and D in Thailand using specific primers designed from species specific sequences. The PCR protocol was optimized to obtain products of 120 bp, 75 bp, 60 bp and 172 bp for species A, B, C and D, respectively. This method used a cocktail of four primer sets to identify these An. dirus sibling species. The method is very sensitive as only a small portion of mosquito was required allowing the rest of the mosquito to be used for other analyses. Specimens also kept for up to 14 years could be analyzed unambiguously from either larvae or adult. This method is advantageous over other PCR-based methods for identification of malaria vectors because it does not require any specific DNA extraction. A mosquito specimen was homogenized in 1x PCR buffer, then the supernatant directly used for PCR identification, allowing a large number of samples to be processed at the same time. It provides a simple and rapid practical method for screening An. dirus species, which is essential in malaria vector epidemiological studies in Southeast Asia.

Field evaluation of simplified radioactive and nonradioactive DNA probe methods for the diagnosis of falciparum malaria.

Specific DNA probe hybridization technique is one method of choice for detection of malaria infection. It provides an obvious advantage over conventional microscopy when large numbers of samples are simultaneously monitored. The method was simplified so that preparation and processing of blood specimens were all performed on membrane filters. Background signals generated from blood components were removed by treating samples spotted on the membrane with a series of buffer washes without the necessity of a protease digestion step. Hybridization was monitored using either 32P-or digoxigenin-labeled DNA probe. 849 field samples collected from various malaria endemic areas in Thailand have been evaluated by this protocol and compared with microscopic examination. Sensitivity obtained by this procedure was comparable to that of microscopy at a malaria clinic. The specificities of both types of DNA probes were better than 93%, but digoxigenin-labeled probe performed better than 32P-labeled one when the numbers of parasites were less than 25 per 200 white blood cells.

A simplified and highly sensitive detection of Trypanosoma evansi by DNA amplification.

In Southeast Asia Trypanosoma evansi infection is a disease of economic importance since it affects the health of buffalo, cattle and swine. The acute stage symptoms include abortion, central nervous system disorder and even death, and in the chronic condition working capacity and productivity of the animals are affected. A polymerase chain reaction (PCR)-based detection technique has been developed with a sensitivity of 0.5 pg of parasite DNA or one single parasite in 10 microliters of blood samples which were allowed to clot and then boiled before DNA amplification. This permitted storage of blood collection at ambient temperature for at least one month. Phosphate-saline-glucose solution, normally used as trypanosome maintenance buffer, inhibited PCR. Although DNA primers used were derived from T. evansi specific sequence, amplification of the genome of T. brucei and T. equiperdum generated the same 227 bp fragment. This method should now make it possible to detect infections in livestock in the very early stages where microscope examination is equivocal and to monitor groups of animals after trypanocidal treatment.

Specific DNA probe for the sensitive detection of Trypanosoma evansi.

Trypanosoma evansi is the parasitic protozoon that causes "Surra", a wasting disease of domestic animals. Detection of T. evansi plays an important role in epidemiology and animal health. DNA probes were constructed from T. evansi genomic DNA and kinetoplast DNA for sensitive detection of the parasite in infected blood. A 6.5 kb DNA insert of pMUT ec6 plasmid derived from the genomic DNA of T. evansi Npl isolate, selected from 575 recombinant E. coli exhibited the strongest nucleic acid hybridization signal to the T. evansi DNA. Using as the DNA probe, pMUT ec6 could detect as little as 60 pg T. evansi DNA and it did not hybridize to the DNA of cattle, waterbuffalo and two related blood parasites. A simple detection procedure by spotting 10 microliters infected blood onto nylon membrane could sense as little as 1000 parasites. The kinetoplast DNA was cloned in E. coli and found to show a comparable sensitivity to that of the pMUT ec6. However, the kinetoplast DNA exhibited variation in copy number among parasite isolates thus pMUT ec6 should be the DNA probe of choice for sensitive detection of T. evansi.

Detection of Plasmodium falciparum using a cloned DNA probe: a simple procedure suitable for field application.

A simple procedure was developed for spotting blood samples directly onto nylon membrane filter, without the necessity to treat samples with pronase or proteinase K, followed by hybridizing with 32P-labelled DNA probe, pUNK1-45. This probe detected specifically P. falciparum DNA and did not cross react with DNA from man, P. knowlesi, P. chabaudi or P. cynomolgi. The probe was sensitive to detect a parasitemia of 0.001% in 20 microliters of blood.

Diagnostic restriction fragment patterns of DNA from the four isomorphic species of Anopheles dirus.

Total DNA from isofemale lines of the four isomorphic species of the Anopheles dirus were screened against twenty restriction endonucleases. Seven enzymes (Ava II, Alu I, Bgl II, Hae III, Hinf I, Mbo I and Sau3A I) produced some unique DNA fragments for each of the lines. Seven other enzymes (BamH I, BstN I, Cfo I, EcoR I, Kpn I, Nru I and Pst I) produced unique fragments in two of the lines. The remaining six enzymes (Hha I, Mnl I, Msp I, Nae I, Rsa I and Taq I) gave vague patterns which might result from either biological or technical causes. The results demonstrated that restriction fragment length of the DNA could be used as a means to distinguish isomorphic species of the Anopheles dirus.