Cost-effectiveness analysis of alkaline lysis, MagNA Pure,and phenol-chloroform DNA extraction methodsfollowed by measurement of single gene copynumber using quantitative real-time PCR forDirofilaria immitis microfilaria
Objective : Dirofilariasis or heartworm disease, a mosquito-borne parasitic disease,continues to be a serious problem in canine (dogs) and feline (cats) species.The disease is caused mainly by the filarial nematode, Dirofilaria immitis. Incanine dirofilariasis, the adult worms, present in the pulmonary arteries andthe right ventricle, causing damages and leading to pulmonary hypertensionand right-sided congestive heart failure. In addition, acute death in felinedirofilariasis causes lung injury resulting in respiratory distress. Being criticalto transmission and complex disease pathology, the microfilarial stage isuseful in laboratory diagnosis; however, the microfilaria identification processis laborious, difficult and time-consuming. Development of a moleculardiagnostic will provide an efficient alternative. The present study analyzesrelative efficiencies of three different methods of DNA extraction from D.immitis microfilariae for quantitative real-time PCR (qPCR).Methods : Three different microfilaria DNA extraction methods: alkaline lysis, MagNAPure and phenol-chloroform were studied. DNA extracted from 1, 10, and100 D. immitis microfilariae were analyzed by qPCR, targeting the singlecopy of filarial glutathione-S-transferase (gst) gene.Results : We were able to detect as low as 0.025-microfilaria by qPCR following analkaline lysis extraction. The alkaline lysis method requires the least amountof time to complete (20 min) and it is also the least expensive, less than$0.05 per sample.Conclusion : The alkaline lysis DNA extraction method appeares to be the most sensitive,least time-consuming, and most cost-effective method for D. immitismicrofilariae. This technique is likely to provide the most suitable platform forimproved detection and measurement of microfilarial copy number and‘free’ filarial DNA. In particular, this technique will advance diagnosis offeline dirofilariasis that involves characteristically low circulating microfilariaeand amicrofilaremia.
Faculty of Medicine, Chulalongkorn University
Sungpradit, S; Nuchprayoon, S; and Chatsuwan, T.
"Cost-effectiveness analysis of alkaline lysis, MagNA Pure,and phenol-chloroform DNA extraction methodsfollowed by measurement of single gene copynumber using quantitative real-time PCR forDirofilaria immitis microfilaria,"
Chulalongkorn Medical Journal: Vol. 54:
6, Article 3.
Available at: https://digital.car.chula.ac.th/clmjournal/vol54/iss6/3