Korean Journal of Clinical Laboratory Science (대한임상검사과학회지)

Korean Society for Clinical Laboratory Science (대한임상검사과학회)
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Development of Real-time PCR Assays for Detection of Dirofilaria immitis from Infected Dog Blood

심장사상충에 감염된 개의 혈액에서 심장사상충 유전자를 검출할 수 있는 실시간 중합효소연쇄반응 기법 개발

  • Oh, In Young (Department of Biomedical Laboratory Sciences, College of Health Science, Eulji University) ;
  • Kim, Kyung Tae (ALPHAGENE Co., Ltd., Singu University, Business Incubation Center) ;
  • Jun, Jin Hyun (Department of Biomedical Laboratory Sciences, College of Health Science, Eulji University) ;
  • Shin, Jae-Ho (Department of Biomedical Laboratory Sciences, College of Health Science, Eulji University) ;
  • Sung, Ho Joong (Department of Biomedical Laboratory Sciences, College of Health Science, Eulji University)
  • Received : 2016.04.21
  • Accepted : 2016.05.13
  • Published : 2016.06.30
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Abstract

Dirofilaria immitis is a filarial nematode parasite that causes cardiopulmonary dirofilariasis in dogs. The purpose of this study was the development of real-time PCR assays for efficient detection of D. immitis. The D. immitis-specific primers confirmed in our previous study and a newly designed TaqMan probe were used for quantitative diagnostics. First, SYBR Green real-time PCR was performed using the specific primers and serially diluted genomic DNA or plasmid DNA, and melting curve analyses were performed after amplification. The melting curve showed one specific peak in each of the genomic and plasmid DNA reactions, suggesting that the primers specifically amplify the D. immitis cytochrome c oxidase subunit I gene. Comparison of SYBR Green and TaqMan real-time PCR using serially diluted plasmid DNA showed higher efficiency and specificity with TaqMan real-time PCR. The real-time PCR assays developed in this study will provide improved diagnostic methods to overcome the limitations of conventional diagnostic tools and facilitate more rapid and accurate diagnoses.

선형 사상충의 일종인 심장사상충은 개의 심폐 사상충증을 유발한다. 이에 본 연구의 목적은 심장사상충을 효과적으로 검출할 수 있는 실시간 중합효소연쇄반응 기법을 개발함에 있다. 연구에 있어서 사용된 프라이머 및 프로브는 선행연구에서 제작된 심장사상충 특이 프라이머 및 새롭게 제작된 TaqMan 프로브를 이용하였다. 선행연구에서 제작된 프라이머 및 농도별로 희석된 게놈유전자와 플라스미드유전자가 SYBR Green 실시간 중합효소연쇄반응 수행에 이용되었으며, 중합효소연쇄반응 과정 중 증폭 이후의 녹는 곡선의 결과를 분석하였다. 분석결과 사용된 프라이머는 각각 게놈유전자 및 플라스미드 유전자에서 특이 녹는 곡선을 나타냄에 따라 심장사상충 특이 사이토크롬 C 산화효소 유전자만을 증폭하고 있음을 확인 할 수 있었다. 새롭게 제작된 TaqMan 프로브는 SYBR Green 실시간 중합효소연쇄반응과의 결과를 농도별로 희석된 플라스미드 유전자를 이용하여 비교 분석하였고, 분석결과 TaqMan 프로브를 이용한 실시간 중합효소연쇄반응이 검출효율 및 특이도에 있어서 우수함을 확인할 수 있었다. 본 연구를 통하여 개발한 실시간 중합효소연쇄반응은 기존의 전통적인 진단기법의 한계를 극복할 수 있는 신속하고 정확한 향상된 진단기법을 제시한다.

Keywords

References

  1. Watts KJ, Courteny CH, Reddy GR. Development of a PCR- and probe-based test for the sensitive and specific detection of the dog heartworm, Dirofilaria immitis, in its mosquito intermediate host. Mol Cell Probes. 1999;13:425-430. https://doi.org/10.1006/mcpr.1999.0270
  2. Kronefeld M, Kampen H, Sassnau R, Werner D. Molecular detection of Dirofilaria immitis, Dirofilaria repens and Setaria tundra in mosquitoes from Germany. Parasit Vectors. 2014;7:30. https://doi.org/10.1186/1756-3305-7-30
  3. Khedri J, Radfar MH, Borji H, Azizzadeh M, Akhtardanesh B. Canine heartworm in southeastern of Iran with review of disease distribution. Iran J Parasitol. 2014;9:560-567.
  4. Fu Y, Lan J, Wu X, Yang D, Zhang Z, Nie H, et al. Expression of translationally controlled tumor protein (TCTP) gene of Dirofilaria immitis guided by transcriptomic screening. Korean J Parasitol. 2014;52:21-26. https://doi.org/10.3347/kjp.2014.52.1.21
  5. Rossi MI, Aguiar-Alves F, Santos S, Paiva J, Bendas A, Fernandes O, et al. Detection of Wolbachia DNA in blood from dogs infected with Dirofilaria immitis. Exp Parasitol. 2010;126:270-272. https://doi.org/10.1016/j.exppara.2010.05.002
  6. Nuchprayoon S, Junpee A, Poovorawan Y, Scott AL. Detection and differentiation of filarial parasites by universal primers and polymerase chain reaction-restriction fragment length polymorphism analysis. Am J Trop Med Hyg. 2005;73:895-900. https://doi.org/10.4269/ajtmh.2005.73.895
  7. Masetti A, Rivasi F, Bellini R. Mosquito-based survey for the detection of flaviviruses and filarial nematodes in Aedes albopictus and other anthropophilic mosquitoes collected in northern Italy. New Microbiol. 2008;31:457-465.
  8. Mazzariol S, Cassini R, Voltan L, Aresu L, Frangipane di Regalbono A. Heartworm (Dirofilaria immitis) infection in a leopard (Panthera pardus pardus) housed in a zoological park in north-eastern Italy. Parasit Vectors. 2010;3:25. https://doi.org/10.1186/1756-3305-3-25
  9. Furtado AP, Do Carmo ES, Giese EG, Vallinoto AC, Lanfredi RM, Santos JN. Detection of dog filariasis in Marajo island, Brazil by classical and molecular methods. Parasitol Res. 2009;105:1509-1515. https://doi.org/10.1007/s00436-009-1584-9
  10. Rojas A, Rojas D, Montenegro VM, Baneth G. Detection of Dirofilaria immitis and other arthropod-borne filarioids by an HRM real-time qPCR, blood-concentrating techniques and a serological assay in dogs from Costa Rica. Parasit Vectors. 2015;8:170. https://doi.org/10.1186/s13071-015-0783-8
  11. Schnyder M, Deplazes P. Cross-reactions of sera from dogs infected with Angiostrongylus vasorum in commercially available Dirofilaria immitis test kits. Parasit Vectors. 2012;5:258. https://doi.org/10.1186/1756-3305-5-258
  12. Oi M, Sato Y, Nakagaki K, Nogami S. Detection of Dirofilaria immitis DNA in host serum by nested PCR. Parasitol Res. 2015;114:3645-3648. https://doi.org/10.1007/s00436-015-4591-z
  13. Hwang IK, Lee HY, Kim MH, Jo HS, Choi DH, Kang PW, et al. Development of real-time PCR assay for genetic identification of the mottled skate, Beringraja pulchra. Forensic Sci Int. 2015;255:80-84. https://doi.org/10.1016/j.forsciint.2015.05.028
  14. Carracedo A, Bar W, Lincoln P, Mayr W, Morling N, Olaisen B, et al. DNA commission of the international society for forensic genetics: guidelines for mitochondrial DNA typing. Forensic Sci Int. 2000;110(2):79-85. https://doi.org/10.1016/S0379-0738(00)00161-4
  15. Dawnay N, Ogden R, McEwing R, Carvalho GR, Thorpe RS. Validation of the barcoding gene COI for use in forensic genetic species identification. Forensic Sci Int. 2007;173(1):1-6. https://doi.org/10.1016/j.forsciint.2006.09.013
  16. Hebert PD, Ratnasingham S, deWaard JR. Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proc Biol Sci. 2003;270(Suppl 1):S96-99.
  17. Hebert PD, Cywinska A, Ball SL, deWaard JR. Biological identifications through DNA barcodes. Proc Biol Sci. 2003;270(1512):313-321. https://doi.org/10.1098/rspb.2002.2218
  18. De Gregorio E, Roscetto E, Iula VD, Martinucci M, Zarrilli R, Di Nocera PP, et al. Development of a real-time PCR assay for the rapid detection of Acinetobacter baumannii from whole blood samples. New Microbiol. 2015;38:251-257.
  19. Liu T, Yang B, Song X, Wang X, Yuan Y, Liu L, et al. Detection and quantification of hepatopancreatic parvovirus in penaeid shrimp by real-time PCR assay. J Invertebr Pathol. 2013;114:309-312. https://doi.org/10.1016/j.jip.2013.09.007
  20. Liu CM, Kachur S, Dwan MG, Abraham AG, Aziz M, Hsueh PR, et al. FungiQuant: a broad-coverage fungal quantitative real-time PCR assay. BMC Microbiol. 2012;12:255. https://doi.org/10.1186/1471-2180-12-255
  21. Li K, Gao H, Gao L, Qi X, Qin L, Gao Y, et al. Development of TaqMan real-time PCR assay for detection and quantitation of reticuloendotheliosis virus. J Virol Methods. 2012;179:402-408. https://doi.org/10.1016/j.jviromet.2011.12.001
  22. Qu S, Shi Q, Zhou L, Guo Z, Zhou D, Zhai J, et al. Ambient stable quantitative PCR reagents for the detection of Yersinia pestis. PLoS Negl Trop Dis. 2010;4:e629. https://doi.org/10.1371/journal.pntd.0000629
  23. Cho JA, Kim DW, Jeong SD, Cheon JS, Na GA, Kim HR, et al. Utility of real time RT-PCR for the quantitative detection of minimal residual disease in hematological malignancy. Korean J Clin Lab Sci. 2009;41:11-23.
  24. Latrofa MS, Montarsi F, Ciocchetta S, Annoscia G, Gantas-Torres F, Ravaqnan S, et al. Molecular xenomonitoring of Dirofilaria immitis and Dirofilaria repens in mosquitoes from north-eastern Italy by real-time PCR coupled with melting curve analysis. Parasite Vectors. 2012;5:76. https://doi.org/10.1186/1756-3305-5-76

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