APPLICATION OF DRIED BLOOD SAMPLE ON FTA® PAPER FOR DETECTION OF TRYPANOSOMA EVANSI BY PCR
2. MATERIALS AND METHODS 1. Parasites
2.4. Polymerase chain reaction (PCR)
Specific oligonucleotide primer used for the amplification of T.evansi DNA, was derived from repetitive nucleotide sequences [15]. The PCR amplification was performed using Thermal Cycler (Hybaid Sprint). Each reaction was carried out in 25 μL volume containing 1x PCR buffer (QAIGEN), 1.5 mM MgCl2, 25 μM of each primer, TR3 (5’-GCGCGGATTCTTTGCAGACGA-3’) and TR4 (5’-TGCAGACAC TGGAATGTTACT-3’) and 100 unit of Taq polymerase (QAIGEN). The PCR mixture was heated at 95oC for 5 min prior to monitoring the PCR cycle. One cycle of PCR was consisted of denaturation at 95oC for 45 s, annealing at 55oC for 45 s and polymerization at 72oC for 45 s. The cycle was proceeded for additional 29 cycles in the Thermal cycler. Ten microlitres of PCR products of approximately 257 bp in size was analysed by using 1.5% agarose gel electrophoresis, ethidium bromide staining and visualized under UV transilluminator.
3. RESULTS
To determine the minimum parasites/mL detected using PCR, DNA templates obtained from 2 different methods was prepared and tested. Results (Fig. 1) showed that PCR could detect parasite DNA at the level as less as 6.0 x10-2 parasites/mL which
DNA samples derived from the method of Chelex-100® preparation, whereas 2.2 x 105 parasites/mL which DNA samples derived from dried blood on the papers.
FIG. 1. Sensitivity of the each individual PCR.
Obtained using DNA templates derived from whole bloods (50 μL). Lane 1, Purified T.evansi DNA; lane 2-3, 6.0 x107 parasites/mL; lane 4-5, 6.0 x105 parasites/mL; lane 6-7, 6.0 x103 parasites/mL; lane 8-9, 6.0 x101 parasites/mL; lane 10-11, 6.0 x10-2 parasites/mL;
lane 12-13, 6.0 x10-4 parasites/mL; lane 14-15, 6.0 x10-6 parasites/mL; lane M contains the molecular weight size markers consisting of fragment 100-1500 bp.
FIG. 2. Sensitivity of the each individual PCR.
DNA templates derived from dried blood on FTA papers: Lane 1, purified T.evansi DNA;
lane 2, 2.2 x105 parasites/mL; lane 3, 2.2 x103 parasites/mL; lane 4, 2.2 x101 parasites/mL;
lane 5, 2.2 x10-2 parasites/mL; lane 6, 2.2 x10-4 parasites/mL; lane 7, 2.2 x10-6 parasites/mL; lane 8, 2.2 x10-8 parasites/mL; lane 9, normal mouse blood; lane M contains the molecular weight size markers consisting of fragment 100-1500 bp.
Table I show that the determination by PCR using DNA templates derived from whole blood was more sensitive to the MHCT and MIT whereas using DNA templates derived from dried blood was equivalent to WBF.
TABLE I. SENSITIVITY OF T.EVANSI DETECTION IN DIFFERENT METHODS
2.2 x
4. DISCUSSION
The sensitivity of the PCR in this study for detection of T.evansi using DNA templates derived from Chelex-100® preparation was 0.022-0.06 parasite/mL which the volume of DNA templates was 5 μL (the equivalent of 100-300 parasites). It was higher than that of previously published PCR assays. The minimal level of detection at 1 pg of T.evansi DNA (corresponded to100 parasites/mL) was reported by using the same primers in this study [16]. Moreover, PCR-ELISA could detect T.evansi DNA at the level as less as 0.01 pg (corresponded to 1 parasite/mL) was also reported by using the same primers in this study [17]. It is likely that the sensitivity of the PCR depends on the volume of blood sample taken prior to DNA template preparation and reduction of PCR inhibitors such as haemoglobin and heavy metals. In this study, haemoglobin was removed by washing twice in 0.1 M Ammonium chloride. Heavy metals were absorbed by Chelex-100®. The result showed that PCR-based assay for the detection of T.evansi present in the dried blood on FTA® Paper was tested in parallel to the present of T.evansi in the whole blood. The sensitivity of this method was equivalent to the conventional WBF although the volume of dried blood on FTA® paper was less than 10 μL.
Haemoglobin, a PCR inhibitor was easily to remove from the papers by washing twice in FTA® Purification Reagent or 0.002% SDS. The sample papers in white colour (approximately 1/4 of paper absorbed 10 μL of blood) were dried up and directly placed into the PCR tube as DNA templates. The main advantage of using dried blood on papers as DNA templates is that the sample can be stored at room temperature and be convenient for shipment via mail. This method is suitable for convenient sample collection and shipment. That is beneficial for epidemiological survey. In addition, it is also useful for designing rational Trypanosomiasis control program in the endemic area.
ACKNOWLEDGEMENTS
This research project number THA/11417, CRP D3.20.21 was supported under Research contract No. 11417/R2 of the International Atomic Energy Agency (IAEA).
REFERENCES
[1] BOONYAWONG, T., CHANTARAPRATEEP, P., MUANGYAI, M. Surra in horse. Thai J. Vet. Med., 5: 665–672 (1975).
[2] MATTHIAS, E., MUANGYAI, M. Trypanosoma evansi infection in a swamp buffaloes calf. Thai J. Vet. Med., 10: 47–54 (1980).
[3] LOEHR, K.F., PHOLPARK, S., SRIKITJAKARN, L. BETTERMAN, G., STAAK, C. Trypanosoma evansi infection in buffaloes in north-east Thailand.
1. Field investigation. Trop. Anim. Hlth. Prod., 17: 121–125 (1985).
[4] LOEHR, K.F., PHOLPARK, S., UPATUM, N., LEIDL, K., STAAK, C.
Trypanosoma evansi infection, a frequent cause of abortion in buffaloes. Trop.
Anim. Health Prod., 18: 103–108 (1986).
[5] TRISANAROM, A., MARKMEE, S., PEDUGSORN, C. Trypanosoma evansi infection in Chiengmai dairy cattle. In: Proc. 6th Ann. Livestock Conf., Dept.
Livestock Development, Thailand, 18-19 May 1987, pp. 1–12 (1987).
[6] CHAICHANAPUNPOL, I., SIRIRANGKAMANONT, T., TRONGWONGSA, L., SUVANWASI, P. Observations on Trypanosomiasis in a native bull.
Kasetsart Vet., 6: 1–9 (1985).
[7] TUNTASUVAN, D., SARATAPHAN, N., NISHIKAWA, H. CNS Trypanosomiasis in native cattle. Vet. Parasitol., 73: 357–363 (1997).
[8] TEERAPRASERT, E., CHAICHANAPUNPOOL, I., TRONGWONGSA, L. A case report of Trypanosoma evansi infection in pigs. Proceedings of the 11th Annual Livestock Conference. Department of Livestock Development, Thailand, 12-14 December 1984, pp. 43–64 (1984).
[9] INDRAKAMHANG, P., MOHKAEW, K., ROONGUTHAI, P. Preliminary report on Trypanosoma sp. in native Sambar deer and imported Rusa deer raised in a deer farm in Thailand. In: Proc. 16th Ann. Livestock Conf., Dept. Livestock Development, Thailand, 3–4 May 1996, pp. 55–67 (1996).
[10] TUNTASUVAN, D., MIMAPAN, S., SARATAPHAN, N., TRONGWONGSA, L., INTRARAKSA, R., LUCKINS, A.G. Detection of Trypanosoma evansi in brains of the naturally infected hog deer by streptavidine-biotin immunohistochemistry. Vet. Parasitol., 87: 223–230 (2000).
[11] BAJYANA SONGA, E., HARMERS-CASTERMAN, C., HAMERS, R. The use of the card agglutination test (TestTryp CATT) for the detection of T.evansi infection: A comparison with other Trypanosomosis diagnostic tests under field conditions in Thailand. Ann. Soc. Belg. Med. Trop., 67: 137–148 (1987).
[12] WILSON, A.J. Value of the indirect fluorescent antibody test as a serological aid to diagnosis of Glossina-transmitted bovine Trypanomiasis. Trop. Anim.
Health. Prod., 3: 133–139 (1969).
[13] TUNTASUVAN, D., CHOMPOOCHAN, T., WONGPAKORN, M., MOHKAEW, K. Development on the detection of Trypanosoma evansi in pigs using enzyme-linked immunosorbent assay. J. Thai Vet. Med. Assoc., 47: 45–53 (1996).
[14] WUYTS, N., CHOKESAJJAWATEE, N., PANYIM, S. A simplified and highly sensitive detection of Trypanosoma evansi by DNA amplification. Southeast Asian J. Trop. Med. Public Health, 25: 266–271 (1994).
[15] WUYTS, N., CHOKESAJJAWATEE, N., SARATAPHAN, N., PANYIM, S.
PCR amplification of crude blood on microscope slides in the diagnosis of Trypanosoma evansi infection in dairy cattle. Annual Society of Belgium Medical Tropics, 75: 229–237 (1995).
[16] TANANYTTHAWONGESE, C., SAENGSOMBAT, K., SUKHUMSIRICHAT, W., UTHAISANG, W., SARATAPHAN, N., CHANSIRI, K. Detection of bovine haemoparasite infection using multiplex polymerase chain reaction.
Science Asia, 25: 85–90 (1999).
[17] CHANSIRI, K., KHUCHAREONTAWON, S., SARATAPHAN, N. PCR-ELISA for diagnosis of Trypanosoma evansi in animals and vector. Mol. Cell. Probes, 16: 173–177 (2002).
DETERMINATION OF THE TRYPANOSOMA CONGOLENSE AND THE