应用高分辨率熔解曲线 qPCR 技术检测和定量粪便样本中的犬旋毛虫
Detection and quantification of Spirocerca lupi by HRM qPCR in fecal samples from dogs with spirocercosis.
机构信息
Koret School of Veterinary Medicine, The Hebrew University of Jerusalem, P.O. Box 12, 7610001, Rehovot, Israel.
Kimron Veterinary Institute, 50250, Bet Dagan, Israel.
出版信息
Parasit Vectors. 2017 Sep 19;10(1):435. doi: 10.1186/s13071-017-2374-3.
BACKGROUND
Spirocerca lupi, the dog oesophageal nematode, causes a potentially fatal disease in domestic dogs, and is currently clinically diagnosed by coproscopy and oesophagoscopy. To date, a single molecular method, a semi-nested PCR, targeting the cox1 gene, has been developed to aid in the diagnosis of spirocercosis. The present study describes three novel high-resolution melt (HRM) quantitative PCR (qPCR) assays targeting fragments of the ITS1, 18S and cytb loci of S. lupi. The performance of these molecular assays in feces was compared to fecal flotation and to the previously described cox1 gene semi-nested PCR in 18 fecal samples from dogs with clinical oesophageal spirocercosis diagnosed by oesophagoscopy.
RESULTS
The HRM qPCR for ITS1 and 18S were both able to detect 0.2 S. lupi eggs per gram (epg), while the HRM qPCR for the cytb and the semi-nested PCR for the cox1 detected 6 epg and 526 epg, respectively. Spirocerca lupi was detected in 61.1%, 44.4%, 27.8%, 11.1% and 5.6% of the fecal samples of dogs diagnosed with spirocercosis by using the ITS1 and 18S HRM qPCR assays, fecal flotation, cytb HRM qPCR and cox1 semi-nested PCR, respectively. All dogs positive by fecal flotation were also positive by ITS1 and 18S HRM qPCRs. Quantification of S. lupi eggs was successfully achieved in the HRM qPCRs and compared to the fecal flotation with no significant difference in the calculated concentrations between the HRM qPCRs that detected the 18S and ITS1 loci and the fecal flotation. The HRM qPCR for the 18S cross-amplified DNA from Toxocara canis and Toxascaris leonina. In contrast, the HRM qPCR for ITS1 did not cross-amplify DNA from other canine gastrointestinal parasites.
CONCLUSIONS
This study presents two new molecular assays with significantly increased sensitivity for confirming and quantifying fecal S. lupi eggs. Of these, the HRM qPCR for ITS1 showed the best performance in terms of the limit of detection and absence of cross-amplification with other parasites. These assays will be useful in detecting infection and for follow-up during therapy.
背景
犬食道线虫（Spirocerca lupi）是一种潜在致命的犬科寄生虫，目前临床上通过粪便检查和食道镜检查进行诊断。迄今为止，已经开发出一种针对cox1 基因的单分子方法，即半巢式 PCR，以辅助螺旋体病的诊断。本研究描述了三种针对 S. lupi ITS1、18S 和 cytb 基因片段的新型高分辨率熔解（HRM）定量 PCR（qPCR）检测方法。在 18 份经食道镜检查确诊为临床食道螺旋体病的犬粪便样本中，比较了这些分子检测方法与粪便漂浮法和先前描述的 cox1 基因半巢式 PCR 的检测效果。
结果
ITS1 和 18S 的 HRM qPCR 均能检测到 0.2 个 S. lupi 卵/克（epg），而 cytb 的 HRM qPCR 和 cox1 基因的半巢式 PCR 分别能检测到 6 epg 和 526 epg。使用 ITS1 和 18S HRM qPCR 检测犬食道螺旋体病粪便样本，分别有 61.1%、44.4%、27.8%、11.1%和 5.6%的犬样本检测到 S. lupi，而粪便漂浮法、cytb HRM qPCR 和 cox1 半巢式 PCR 的阳性率分别为 11.1%、5.6%和 0%。所有经粪便漂浮法检测为阳性的犬样本，其 ITS1 和 18S HRM qPCR 检测也均为阳性。HRM qPCR 能够成功地对 S. lupi 卵进行定量，并与粪便漂浮法进行比较，在检测 18S 和 ITS1 基因座的 HRM qPCR 与粪便漂浮法之间，计算浓度无显著差异。Toxocara canis 和 Toxascaris leonina 的 18S HRM qPCR 也可以扩增 DNA，但 ITS1 HRM qPCR 不能扩增其他犬科胃肠道寄生虫的 DNA。
结论
本研究提出了两种新的分子检测方法，具有显著提高的灵敏度，可用于确认和定量粪便中 S. lupi 卵。其中，ITS1 HRM qPCR 在检测限和与其他寄生虫的交叉扩增方面表现最佳。这些检测方法将有助于检测感染，并在治疗过程中进行随访。
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