比较针对不同 DNA 区域的多重平行实时定量 PCR 检测粪便中土源性蠕虫。
Comparison of multi-parallel quantitative real-time PCRs targeting different DNA regions and detecting soil-transmitted helminths in stool.
机构信息
Department of Biological Sciences, Smith College, Northampton, MA, 01063, USA.
Biodiversity & Health, Natural History Museum, Cromwell Road, London, SW7 5BD, UK.
出版信息
Parasit Vectors. 2024 Sep 13;17(1):390. doi: 10.1186/s13071-024-06464-6.
BACKGROUND
Soil-transmitted helminths infect an estimated 18% of the world's population, causing a significant health burden. Microscopy has been the primary tool for diagnosing eggs from fecal samples, but its sensitivity drops in low-prevalence settings. Quantitative real-time polymerase chain reaction (qPCR) is slowly increasing in research and clinical settings. However, there is still no consensus on preferred qPCR targets.
METHODS
We aimed to compare soil-transmitted helminth (STH) DNA detection methods by testing naïve stool samples spiked with known quantities of STH eggs and larvae. DNA extracts from spiked samples were tested using independent quantitative realtime PCR (qPCR) assays targeting ribosomal or putative non-protein coding satellite sequences.
RESULTS
For Trichuris trichiura, there was a strong correlation between egg/larvae counts and qPCR results using either qPCR method (0.86 and 0.87, respectively). Strong correlations also existed for A. lumbricoides (0.60 and 0.63, respectively), but weaker correlations were found for Ancylostoma duodenale (0.41 for both assays) and Strongyloides stercoralis (0.48 and 0.65, respectively). No correlation for Necator americanus was observed when testing with either qPCR assay. Both assays had fair-to-moderate agreement across targets when using field-collected stool samples (0.28-0.45, for all STHs), except for S. stercoralis (0.12) with slight agreement.
CONCLUSIONS
There is a strong correlation between qPCR results and egg/larvae counts. Our study confirms that qPCR is an effective diagnostic tool, even with low-intensity infections, regardless of the DNA-based diagnostic marker used. However, the moderate agreement between the two different qPCR assays when testing field samples highlights the need to understand the role of these targets in the genome so that the parasite burden can be quantified more accurately and consistently by qPCR.
背景
土壤传播性蠕虫感染估计占世界人口的 18%，造成了巨大的健康负担。显微镜检查一直是从粪便样本中诊断虫卵的主要工具，但在低流行地区其敏感性下降。实时定量聚合酶链反应（qPCR）在研究和临床环境中逐渐增加。然而，对于首选的 qPCR 靶点仍然没有共识。
方法
我们旨在通过测试用已知数量的土壤传播性蠕虫卵和幼虫接种的未处理粪便样本，比较土壤传播性蠕虫（STH）DNA 检测方法。从接种样本中提取的 DNA 用针对核糖体或假定非蛋白质编码卫星序列的独立实时定量 PCR（qPCR）检测。
结果
对于 Trichuris trichiura，使用这两种 qPCR 方法时，卵/幼虫计数与 qPCR 结果之间存在很强的相关性（分别为 0.86 和 0.87）。A. lumbricoides 也存在很强的相关性（分别为 0.60 和 0.63），但 Ancylostoma duodenale 的相关性较弱（两种检测方法均为 0.41），Strongyloides stercoralis 的相关性较弱（分别为 0.48 和 0.65）。当使用两种 qPCR 检测时，未检测到 Necator americanus 的相关性。使用现场采集的粪便样本时，两种检测方法在所有 STH 检测中都具有良好到中度的一致性（0.28-0.45），除了 S. stercoralis 为轻度一致性（0.12）。
结论
qPCR 结果与卵/幼虫计数之间存在很强的相关性。我们的研究证实，qPCR 是一种有效的诊断工具，即使在感染强度较低的情况下，也无需考虑使用的 DNA 诊断标志物。然而，当测试现场样本时，两种不同 qPCR 检测方法之间的中等一致性突出表明需要了解这些靶点在基因组中的作用，以便更准确和一致地通过 qPCR 量化寄生虫负担。
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