Generic qPCR assays for quality control in environmental DNA research

This study explored the application of ddPCR and qPCR methods in identifying Naegleria fowleri from both clinical and environmental water samples. Strong agreement between ddPCR and qPCR methods over clinical DNA samples was observed. Naegleria fowleri was present in surface water samples from Lake Pontchartrain during our study period. The ability of N. fowleri to survive in brackish water is therefore a potential risk factor for people who engage in water-related recreational activities. The ddPCR performance demonstrated in this study on clinical and environmental samples lead to greater confidence of ddPCR technology on field application.

To establish the method for extracting exogenous short DNA fragments of Schistosoma japonicum from urine samples, and to evaluate the efficiency of this method for extraction from urine samples treated with various methods. The S. japonicum SjG28 gene fragment was selected as a target sequence, and the 81 bp short DNA fragment was amplified on the target sequence using PCR assay. Following characterization using sequencing, the short DNA fragment was added into the urine samples as an exogenous short DNA fragment. Primers and probes were designed with SjG28 as a target gene, to establish the real-time fluorescent quantitative PCR (qPCR) assay. The sensitivity of this qPCR assay was evaluated with exogenous short DNA fragments that were diluted at a 1:10 dilution ratio as the DNA template, and the specificity of the qPCR assay was evaluated with the genomic DNA of S. mansoni, S. haematobium, Babesia, Ancyiostoma duodenaie, Cionorchis sinensis, and Paragonimus westermani as DNA templates. Exogenous short DNA fragments were added into artificial and healthy volunteers' urine samples, followed by pH adjustment, centrifugation and concentration, and the efficiency of extracting exogenous short DNA fragments from urine samples was compared with the QIAmp Viral RNA Mini Kit (Qiagen kit) and BIOG cfDNA easy kit (BIOG kit). An 81 bp small DNA fragment of S. japonicum was successfully prepared, and the lowest detection limit of the established qPCR assay was 100 copies/μL of the 81 bp small DNA fragment of S. japonicum. If the genomic DNA of S. japonicum, S. mansoni, S. haematobium, Babesia, A. duodenaie, C. sinensis, and P. westermani served as DNA templates, the qPCR assay only detected fluorescent signals with S. japonicum genomic DNA as the DNA template. If the pH values of artificial urine samples were adjusted to 5, 6, 7 and 8, the recovery rates were (49.12 ± 2.09)%, (84.52 ± 4.96)%, (89.38 ± 3.32)% and (87.82 ± 3.90)% for extracting the exogenous short DNA fragment of S. japonicum with the Qiagen kit, and were (2.30 ± 0.07)%, (8.11% ± 0.26)%, (13.35 ± 0.61)% and (20.82 ± 0.68)% with the BIOG kit, respectively (t = 38.702, 26.955, 39.042 and 29.571; all P values < 0.01). If the Qiagen kit was used for extracting the exogenous short DNA fragment from artificial urine samples, the lowest recovery rate was seen from urine samples with a pH value of 5 (all P values < 0.05), and there were no significant differences in the recovery rate from urine samples with pH values of 6, 7 and 8 (all P values > 0.05). Following centrifugation of artificial [(64.30 ± 1.00)% vs. (58.87 ± 0.26)%; t = 12.033, P < 0.05] and healthy volunteers' urine samples [(31 165 ± 1 017) copies/μL vs. (28 471 ± 818) copies/μL; t = 23.164, P < 0.05]. In addition, concentration of artificial urine samples with the 10 kDa Centrifugal Filter and concentration of healthy volunteers' urine samples with the 100 kDa Centrifugal Filter were both effective to increase the recovery of the Qiagen kit for extracting the exogenous short DNA fragment of S. japonicum (both P values < 0.01). A method for extracting exogenous short DNA fragments of S. japonicum from urine samples has been successfully established, and the Qiagen kit has a high extraction efficiency. Adjustment of urine pH to 6 to 8 and concentration of healthy volunteers' urine samples with the 100 kDa Centrifugal Filter are both effective to increase the efficiency of extracting exogenous short DNA fragments of S. japonicum.

Species-specific environmental DNA (eDNA) surveys are increasingly being used to infer species presence in an environment. Current inadequacies in quality control increase concern for false negatives, which can have serious ramifications for both the management of invasive species and the conservation of native species. eDNA surveys involve a multi-step process to sample, capture, extract and amplify target DNA from the environment. We outline various positive control options and show that many of the commonly used controls are capable of detecting false negatives arising during the amplification stage only. We suggest a secondary, generic primer, designed to co-amplify endogenous DNA sampled during species-specific eDNA surveys, constitutes a superior positive control to monitor method success throughout all stages of eDNA analysis. We develop a species-specific European carp (Cyprinus carpio) assay and a generic fish assay for use as an endogenous control for eDNA surveys in Australian freshwater systems where fish are known to be abundant. We use these assays in a multiplex on eDNA samples that are simultaneously sampled, captured, extracted and amplified. This positive control allows us to distinguish method error from informative non-amplification results, improving reliability in eDNA surveys, which will ultimately lead to better informed conservation management decisions.

Background Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Trichomonas vaginalis (TV) cause the three most prevalent sexually transmitted infectious diseases in developed countries, contributing to approximately 300 million new infections worldwide, annually. Due to the challenges of culturing and using immunoassays to detect these pathogens, DNA-based molecular tests have been instrumental, and current trends in molecular testing for CT/NG/TV have moved toward point-of-care tests. Lyophilization provides benefits required for point-of-care tests, such as ambient storage, extended shelf life, simplified downstream workflow, and variable formats. A lyophilized-bead format enables an assay to be placed in sealed consumables and stored at room temperature for up to two years. An additional consideration is the need for endogenous and exogenous controls. Exogenous in-process controls can be used to validate the performance of workflows culminating in a qPCR reaction and provide flexibility to test developers. An ideal exogenous in-process control is nonhomologous to human or human pathogen genome sequences and does not align with primers or probes designed for pathogen detection. The objective of the current study was to develop a lyophilized-bead-format assay containing an ideal exogenous control and establish whether it is a valid option for the accurate detection of CT, NG, and TV. Methods A synthetic DNA control containing targets for CT/NG/TV/RNase P (RP)/exogenous control was used to establish an analytical LOD by qPCR. The 5-plex assay, lyophilized in beads, was hydrated and tested for linearity over an 8-log range of the synthetic DNA control. Further, the qPCR lyophilized bead with assay was tested with a proficiency sample by extracting DNA from ZeptoMetrix NATtrol CT/NG/TV Positive Control and spiking-in the exogenous DNA control. qPCR was performed on the extracted DNA and human cDNA. Results The analytical LOD for the control DNA was determined to be 5 copies/reaction. Additionally, linearity was observed by qPCR over an 8-log range of the synthetic DNA control for all five targets in multiplex with good efficiencies and R2 values. Finally, all targets were amplified and detected by qPCR using DNA extracted from ZeptoMetrix NATtrol CT/NG/TV Positive Control with spiked-in exogenous DNA control. The detection of the exogenous sequence in the qPCR multiplex reaction indicated the extraction was successful. Conclusions We have developed a multiplex qPCR assay in lyophilized-bead format that can identify infectious agents, such as CT/NG/TV, in a single reaction. We have included RP as a human host control, plus an exogenous in-process control, for a total of 5-plex in separate fluorescent channels. We have shown that our lyophilized beads with assay produce a positive qPCR result when combined with a proficiency sample.

Aquatic environmental DNA (eDNA) surveys are sometimes impacted by polymerase chain reaction (PCR) inhibitors. We tested varying concentrations of different inhibitors (humic, phytic, and tannic acids; crude leaf extracts) for impacts on quantitative PCR (qPCR) assays designed for eDNA surveys of bighead and silver carp (Hypophthalmichthys nobilis and Hypophthalmichthys molitrix). We also tested for inhibition by high concentrations of exogenous DNA, hypothesizing that DNA from increasingly closely related species would be increasingly inhibitory. All tested inhibitors impacted qPCR, though only at very high concentrations — likely a function, in part, of having used an inhibitor-resistant qPCR solution. Closer phylogenetic relatedness resulted in inhibition at lower exogenous DNA concentrations, but not at relatively close phylogenetic scales. Inhibition was also influenced by the qPCR reporter dye used. Importantly, different qPCR assays responded differently to the same inhibitor concentrations. Implications of these results are that the inclusion of more than one assay for the same target taxa in an eDNA survey may be an important countermeasure against false negatives and that internal positive controls may not, in the absence of efforts to maximize inhibition compatibility, provide useful information about the inhibition of an eDNA assay.

Aquatic environmental DNA (eDNA) surveys are sometimes impacted by polymerase chain reaction (PCR) inhibitors. We tested varying concentrations of different inhibitors (humic, phytic, and tannic acids; crude leaf extracts) for impacts on quantitative PCR (qPCR) assays designed for eDNA surveys of bighead and silver carp (Hypophthalmichthys nobilis and Hypophthalmichthys molitrix). We also tested for inhibition by high concentrations of exogenous DNA, hypothesizing that DNA from increasingly closely related species would be increasingly inhibitory. All tested inhibitors impacted qPCR, though only at very high concentrations — likely a function, in part, of having used an inhibitor-resistant qPCR solution. Closer phylogenetic relatedness resulted in inhibition at lower exogenous DNA concentrations, but not at relatively close phylogenetic scales. Inhibition was also influenced by the qPCR reporter dye used. Importantly, different qPCR assays responded differently to the same inhibitor concentrations. Implications of these results are that the inclusion of more than one assay for the same target taxa in an eDNA survey may be an important countermeasure against false negatives and that internal positive controls may not, in the absence of efforts to maximize inhibition compatibility, provide useful information about the inhibition of an eDNA assay.

A highly specific Escherichia coli qPCR and its comparison with existing methods for environmental waters

Evaluation of the diagnostic performance of a commercial molecular assay for the screening of suspected monkeypox cases in Sri Lanka

Diet composition and preference of juvenile Murray cod, trout cod and Macquarie perch (Percichthyidae) reared in fertilised earthen ponds

Murray cod, Maccullochella peelii (Mitchell) is an iconic Australian species endemic to the Murray‐Darling Basin (MDB) of inland south‐eastern Australia. Murray cod has been a valuable food source and supported a large commercial fishery throughout much of the 20th century. Over‐fishing and habitat destruction have resulted in significant declines in Murray cod populations throughout much of its range. Since the early 1980s, large numbers of Murray cod have been stocked into waterways to support both recreational fishing and conservation efforts. In this study, the likely impacts of past and current stocking practices on genetic diversity of Murray cod were modelled and new strategies to maximise genetic diversity in stocked populations are explored. The results suggest that a large, well‐managed breeding and stocking programme could help maintain genetic diversity of Murray cod across the MDB. In catchments within the MDB where the effective population size is very small, a well‐designed stocking programme, following strict guidelines for numbers of families reared and number of individuals maintained per family, could increase genetic diversity in a few generations.

Quantification of Escherichia coli O157:H7 in soils using an inhibitor-resistant NanoGene assay

Quantitative PCR (qPCR) assays targeting the host-specific Bacteroides-Prevotella 16S rRNA genetic markers have been proposed as one of the promising approaches to identify the source of fecal contamination in environmental waters. One of the concerns of qPCR assays to environmental samples is the reliability of quantified values, since DNA extraction followed by qPCR assays are usually performed without appropriate sample process control (SPC) and internal amplification controls (IACs). To check the errors in sample processing and improve the reliability of qPCR results, it is essential to evaluate the DNA recovery efficiency and PCR amplification efficiency of the target genetic markers and correct the measurement results. In this study, we constructed a genetically-engineered Escherichia coli K12 strain (designated as strain MG1655 Δlac::kan) as sample process control and evaluated the applicability to environmental water samples. The recovery efficiency of the SPC strain MG1655 Δlac::kan was similar to that of Bacteroides fragilis JCM 11019, when DNA were extracted from water samples spiked with the two bacteria. Furthermore, the SPC was included in the qPCR assays with propidium monoazide (PMA) treatment, which can exclude the genetic markers from dead cells. No significant DNA loss was observed in the PMA treatment. The inclusion of both the SPC (strain MG1655 Δlac::kan) and IAC in qPCR assays with PMA treatment gave the assurance of reliable results of host-specific Bacteroides-Prevotella 16S rRNA genetic markers in environmental water samples.

To evaluate three non-invasive assays for the diagnosis of schistosomiasis mansoni in an Egyptian village. Urine was collected for the detection of circulating cathodic antigen (CCA) and cell-free parasite DNA (cfpd) by Point-of-contact (POC)-cassette assay and PCR, respectively. These tests were compared to Kato-Katz (KK) faecal thick smear for detection of Schistosoma mansoni eggs. Disease prevalence by POC-CCA assay was 86%; by PCR it was 39% vs. 27% by KK. Compared to KK, the sensitivity of POC-CCA reached 100%, but its specificity was only 19.2% with 41% accuracy. Sensitivity of the PCR assay for cfpd was 55.56%, and specificity was 67.12% with 64% accuracy. A new end point was calculated for combined analysis of KK, POC-CCA assay and PCR. Sensitivity for the three tests was 52.94%, 90.2% and 76.47%; specificity was 100% for KK and PCR and 18.37% for POC-CCA. The accuracy calculated for the three tests at the end point was 76% for KK, 55% for POC-CCA assay and 88% for PCR. Conventional PCR assay for detection of cfpd provides a potential screening tool for intestinal schistosomiasis with reliable specificity, reasonable accuracy and affordable financial and technical cost.

Several nucleic acid-based assays have been developed for detecting Anaplasma marginale and Anaplasma centrale in vectors and hosts, making the choice of method to use in endemic areas difficult. We evaluated the ability of the reverse line blot (RLB) hybridisation assay, two nested polymerase chain reaction (nPCR) assays and a duplex real-time quantitative polymerase chain reaction (qPCR) assay to detect A. marginale and A. centrale infections in cattle (n = 66) in South Africa. The lowest detection limits for A. marginale plasmid DNA were 2500 copies by the RLB assay, 250 copies by the nPCR and qPCR assays and 2500, 250 and 25 copies of A. centrale plasmid DNA by the RLB, nPCR and qPCR assays respectively. The qPCR assay detected more A. marginale- and A. centrale-positive samples than the other assays, either as single or mixed infections. Although the results of the qPCR and nPCR tests were in agreement for the majority (38) of A. marginale-positive samples, 13 samples tested negative for A. marginale using nPCR but positive using qPCR. To explain this discrepancy, the target sequence region of the nPCR assay was evaluated by cloning and sequencing the msp1β gene from selected field samples. The results indicated sequence variation in the internal forward primer (AM100) area amongst the South African A. marginale msp1β sequences, resulting in false negatives. We propose the use of the duplex qPCR assay in future studies as it is more sensitive and offers the benefits of quantification and multiplex detection of both Anaplasma spp.

To evaluate routine use of the NeoPath AutoPap 300 QC System (AP 300) as it influences diagnostic quality and operations in a large cytology laboratory. During a three-month period, 35,143 conventionally prepared consecutive cervical cytologic smears taken from non-high-risk women and evaluated as negative by our staff of 25 cytotechnologists were selected for processing by four AP 300 instruments. Slides flagged for review by the AP 300 were reevaluated by our five quality control (QC) cytotechnologists. False negative (FN) results were compared with results of our current practice (CP), random-selection QC method, used during the preceding six months. A 240% increase was seen in the FN detection rate for atypical squamous cells of undetermined significance (ASCUS) and squamous intraepithelial lesions (SIL) (n AP = 65 FN in 5,034 QC, n CP = 77 FN in 22,052 QC) and a 744% increase in the FN detection rate for low and high grade SIL (n AP = 24 FN/5,034 QC, n CP = 12 FN/22,052 QC). The rate of overcall by cytotechnologists did not increase. The QC ASCUS/SIL ratio improved. FN biopsy correlation increased from 45% to 85% (n CP = 17/38 agreement, n AP = 23/27 agreement). Turnaround time increased by one or more days for negative and 1.5 days for QC result reporting. Sensitivity varied among instruments. More FN results and greater specificity were seen using the AP 300 than using CP. As with other instrumentation, each laboratory should establish acceptable ranges of performance and baseline values for sensitivity and specificity.