Cost-effectiveness analysis of alkaline lysis, MagNA Pure, and phenol-chloroform DNA extraction methods followed by measurement of single gene copy number using quantitative real-time PCR for Dirofilaria immitis microfilaria

Abstract

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. In canine dirofilariasis, the adult worms, present in the pulmonary arteries and the right ventricle, causing damages and leading to pulmonary hypertension and right-sided congestive heart failure. In addition, acute death in feline dirofilariasis causes lung injury resulting in respiratory distress. Being critical to transmission and complex disease pathology, the microfilarial stage is useful in laboratory diagnosis; however, the microfilaria identification process is laborious, difficult and time-consuming. Development of a molecular diagnostic will provide an efficient alternative. The present study analyzes relative 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, MagNA Pure and phenol-chloroform were studied. DNA extracted from 1, 10, and 100 D. immitis microfilariae were analyzed by qPCR, targeting the single copy of filarial glutathione-S-transferase (gst) gene.

Results : We were able to detect as low as 0.025-microfilaria by qPCR following an alkaline lysis extraction. The alkaline lysis method requires the least amount of 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. immitis microfilariae. This technique is likely to provide the most suitable platform for improved detection and measurement of microfilarial copy number and ‘free’ filarial DNA. In particular, this technique will advance diagnosis of feline dirofilariasis that involves characteristically low circulating microfilariae and amicrofilaremia.