While waters might be contaminated by oocysts from >40 Cryptosporidium species, only viable oocysts of C. parvum and C. hominis truly pose the main health risk to the immunocompetent population. Oocyst viability is also an important but often neglected risk factor in monitoring waterborne parasites. However, commonly used methods in water monitoring and surveys cannot distinguish species (microscopic observation) or oocyst viability (PCR), as dead oocysts in water could retain gross structure and DNA content for weeks to months. Here, we report new TaqMan qRT-PCR/qPCR assays for quantitative detection of viable C. parvum and C. hominis oocysts. By targeting a hypothetical protein-encoding gene cgd6_3920 that is highly expressed in oocysts and variable between species, the qRT-PCR/qPCR assays achieve excellent analytical specificity and sensitivity (limit of quantification [LOQ] = 0.25 and 1.0 oocyst/reaction). Using calibration curves, the number and ratio of viable oocysts in specimens could be calculated. Additionally, we also establish a TaqMan-18S qPCR for cost-effective screening of pan-Cryptosporidium-positive specimens (LOQ = 0.1 oocyst/reaction). The assay feasibility is validated using field water (N = 43) and soil (79) specimens from 17 locations in Changchun, China, which detects four Cryptosporidium species from seven locations, including three gp60-subtypes (i.e., IIdA19G1, IIdA17G1 and IIdA24G2) of C. parvum oocysts showing varied viability ratios. These new TaqMan q(RT)-PCR assays supplement current methods in the survey of waters and other samples (e.g., surfaces, foods and beverages), and are applicable to assessing the efficiency of oocyst deactivation protocols.

Mammalian orthoreovirus (MRV) infections are ubiquitous in multiple mammalian species including humans, and mainly causes gastroenteritis and respiratory disease. In this study, we developed a rapid and sensitive TaqMan qRT-PCR method for MRV detection based on the primers and probe designed within the conserved L1 gene. The qRT-PCR assay was evaluated for its sensitivity, specificity, efficiency and reproducibility. It was found that the detection sensitivity was equivalent to 10 DNA copies/μL, and the standard curves had a linear correlation of R2 = 0.998 with an amplification efficiency of 99.6%. The inter- and intra-assay coefficients of variation (CV%) were in the range of 0.29% to 2.16% and 1.60% to 3.60%, respectively. The primer sets specifically amplified their respective MRV segments and had the highest detection sensitivities of 100.25 TCID50/mL with amplification efficiencies of 99.5% (R2 = 0.999). qRT-PCR was used for MRV detection from samples of sheep, goats, and calves from four regions in China, and the overall MRV prevalence was 8.2% (35/429), whereas 17/429 (4.0%) were detected by RT-PCR and 14/429 (3.3%) by virus isolation. The qRT-PCR assay showed significantly higher sensitivity than RT-PCR and virus isolation. Results from an epidemiological survey indicated that the positive rate of MRV in rectal swabs from sheep and goats tested in Shaanxi, Jiangsu, and Xinjiang were 9/80 (11.3%), 12/93 (12.9%) and 14/128 (10.9%), respectively. In goats and sheep, MRV prevalence was obviously associated with season and age, with a high positive rate of more than 8% during September to April and approximately 13% in small ruminant animals under two months of age. This is the first instance of MRV infection in sheep and goats in China, thus broadening our knowledge of MRV hosts. Consequently, primer optimization for qRT-PCR should not only prioritize amplification efficiency and specificity, but also sensitivity. This assay will contribute to more accurate and rapid MRV monitoring by epidemiological investigation, viral load, and vaccination efficacy.

Cellular RNAs are often expressed as multiple isoforms of complex heterogeneity in both length and terminal sequences. IsomiRs, the isoforms of microRNAs, are such an example. Distinct quantification of each RNA variant is necessary to unravel the biogenesis mechanism and biological significance of heterogenetic RNA expression. Here we describe Dumbbell-PCR (Db-PCR), a TaqMan RT-PCR-based method that distinctively quantifies a specific small RNA variant with single-nucleotide resolution at terminal sequences. Db-PCR enables the quantitative analysis of RNA terminal heterogeneity without performing Next-Generation Sequencing.