Sensitive detection of specific cell-free DNA in serum samples from sheep with cystic echinococcosis

Abstract

BackgroundDeveloping more sensitive methods for the diagnosis of echinococcosis is essential. In this study PCR assay for sensitive detection of specific cell-free DNA (cfDNA) of Echinococcus granulosusin sensu lato in the sera of the sheep naturally infected with echinococcosis was investigated.MethodsTo extract cfDNA from 35 infected sheep, the modified phenol-chloroform method was used for two different volumes (0.5 and 2 ml) of serum samples. From each extracted sample, two DNA volumes (5 and 10 mu l) were amplified using both standard PCR and semi-nested PCR targeting NADH dehydrogenase subunit I.ResultsStandard and semi-nested PCR on 0.5 ml of serum samples detected Echinococcus DNA in 8 and 12 out of 35 sheep, respectively; however, using 2 ml of serum samples, they detected 24 and 27 samples. By increasing the volume of template DNA, the PCRs could detect 29 and 33 out of 35 samples. The results were confirmed by sequencing of randomly selected PCR amplicons and comparing them with GenBank databases.ConclusionsLarger volumes of serum for DNA extraction, greater volumes of DNA template for PCR, and employing a semi-nested PCR protocol, increased the sensitivity of PCR to 95. This approach can also be applied to the diagnosis of echinococcosis in humans. Cystic echinococcosis (CE) is a disease caused by the larvae of the worm Echinococcus granulosus. Ingesting the eggs of these worms which are excreted in the feces of infected dogs, can result in the formation of cysts in the liver or lungs of humans or herbivorous animals. Although ultrasound is commonly employed for diagnosing these cysts, hydatid cysts can frequently be misdiagnosed as cystic lesions of different origins. Hence, the necessity for faster, more accurate, and non-invasive diagnostic methods is evident. In this study, serum samples were collected from 35 sheep with hydatid cysts, and DNA was extracted and purified. Specific primers were then utilized to amplify the free DNA of the sera via polymerase chain reaction (PCR). Our findings indicate that the diagnostic sensitivity of this disease can be enhanced up to 95 by increasing the volume of serum, employing larger amounts of DNA template in the PCR reaction, and utilizing nested PCR assay. If applied to human samples, this method holds great promise for significantly improving the sensitivity and accuracy of CE diagnosis.