To further ensure the quality of detection, selected individual learn more or pooled PCR products were also sequenced to validate their identities. When the qPCR detection system was used, melting curve analysis was performed to confirm that PCR amplicons showed the same curves as those of positive controls. Among the 54 fecal DNA specimens, we have detected 21 (38.9%) positive samples. However, because the specimens were collected from both individual and pooled quail samples derived from 88 birds, direct calculation of positive rate (i.e., 21/54 = 38.9%) was inappropriate as
pooled positive specimens might selleck compound contain both positive and negative samples. Therefore, we employed two additional approaches to estimate the prevalence. The first approach was to only calculate
positive rate from the 39 samples collected from individual birds (non-pooled), in which 13 samples were positive, giving a 33.3% positive rate. The second approach employed software written by Dr. Brad Biggerstaff as an Excel Add-In (PooledInfRate, version 3.0) (http://www.cdc.gov/westnile/resourcepages/mosqSurvSoft.html), which was originally developed to determine positive rates of viral infections in pooled mosquito samples using a maximum likelihood estimation (MLE) algorithm [22]. By applying a bias-corrected MLE estimation, we obtained CH5183284 order an infection rate of 27.7% with lower and upper limits at 18.6% and 38.7%, respectively (95% confidence interval). Collectively, we conclude that ~30% (or between 28% – 33%) of the sampled wild quail were infected by the eye worm. The actual rate might be even higher, as the fecal
samples were only collected once, rather than continuously Teicoplanin for several days, and the sensitivity of PCR detection might not be maximal due to the inhibitory substances commonly present in fecal samples as discussed below. The detection of O. petrowi DNA in feces allows rapid and sensitive detection of the presence of eye worms without the need to examine individual birds. However, one needs to be aware of the presence of inhibitory substances in fecal samples and the difficulties in releasing DNA from eggs or encysted larvae. The presence of inhibitory substances could be minimized (if not completely eliminated) using the tablets included in the DNA isolation kits specifically designed for stool samples such as the QIAamp DNA Stool Mini Kit (Qiagen). Freeze/thaw cycles combined with homogenization with glass beads were necessary to break the eggs or encysted larvae to ensure the release of DNA. Furthermore, nested PCR might also be used by the addition of a primary amplification using the external primers QEW_2373F and QEW_2681R to not only improve the sensitivity of PCR detection, but to also further eliminate the presence of inhibitory substances in a second amplification procedure. The life cycle of O. petrowi is not well understood, its exact intermediate host(s) as well as its migration details in quail. The presence of O.