Development of an RNA Extraction Protocol for a Norovirus from Raw Oysters and Detection by qRT-PCR and Droplet-Digital RT-PCR

Foodborne viruses such as norovirus and hepatitis A virus cause frequent outbreaks associated with the consumption of raw or undercooked oysters. Viral particles are bioaccumulated in the oyster’s digestive glands, making RNA extraction and RT-PCR detection difficult due to the complex nature of the food matrix and the presence of RT-PCR inhibitors. Herein, we have developed a viral RNA extraction protocol from raw oysters using murine norovirus (MNV) as a surrogate for human noroviruses. The method combines lysis in Tri-Reagent reagent, followed by RNA extraction using Direct-Zol purification columns and lithium chloride precipitation. Viral load quantification was performed by both qRT-PCR and droplet-digital RT-PCR. We have demonstrated that this method can efficiently remove RT-PCR inhibitors, and is sensitive enough to reliably detect viral contamination at 25 PFU/0.2 g. We have also compared the efficiency of this method with the ISO 15216-1:2017 method and Method E developed by Quang and colleagues, and observed significantly higher efficiency compared with the ISO 15216-1 method and comparable efficiency with Method E, with less steps, and shorter hands-on time.

Evaluation of DNA extraction methods for quantifying Helicobacter pylori in water with PCR inhibitors

DNA extraction methods were evaluated to reduce PCR inhibitors and quantify Helicobacter pylori directly from water samples using real-time PCR. Three nucleic acid extraction methods were evaluated for different types of water samples. While the QIAamp DNA mini kit for tissue was suitable for DNA extraction from treated water, the QIAamp DNA stool mini kit was still efficient in analyzing samples from river water after heavy rain and with high concentration of PCR inhibitors. The FastDNA SPIN Kit for Soil could extract DNA effectively from microbes in river and stream waters without heavy rain. Immunomagnetic separation (IMS) was used prior to DNA extraction and was a useful tool for reducing PCR inhibitors in influent and stream samples. H. pylori in various waters could be quantified directly by real-time PCR while minimizing the effect of PCR inhibitors by an appropriate method through the evaluation of DNA extraction methods considering the characteristics of the matrix water. The findings of the present study suggest that the types or characteristics of water sample by source and precipitation are an important factor in detecting H. pylori and they can be applied when detecting and monitoring of other pathogens in water.

Comparing Quantitative Methods for Analyzing Sediment DNA Records of Cyanobacteria in Experimental and Reference Lakes

Sediment DNA (sedDNA) analyses are rapidly emerging as powerful tools for the reconstruction of environmental and evolutionary change. While there are an increasing number of studies using molecular genetic approaches to track changes over time, few studies have compared the coherence between quantitative polymerase chain reaction (PCR) methods and metabarcoding techniques. Primer specificity, bioinformatic analyses, and PCR inhibitors in sediments could affect the quantitative data obtained from these approaches. We compared the performance of droplet digital polymerase chain reaction (ddPCR) and high-throughput sequencing (HTS) for the quantification of target genes of cyanobacteria in lake sediments and tested whether the two techniques similarly reveal expected patterns through time. Absolute concentrations of cyanobacterial 16S rRNA genes were compared between ddPCR and HTS using dated sediment cores collected from two experimental (Lake 227, fertilized since 1969 and Lake 223, acidified from 1976 to 1983) and two reference lakes (Lakes 224 and 442) in the Experimental Lakes Area (ELA), Canada. Relative abundances of Microcystis 16S rRNA (MICR) genes were also compared between the two methods. Moderate to strong positive correlations were found between the molecular approaches among all four cores but results from ddPCR were more consistent with the known history of lake manipulations. A 100-fold increase in ddPCR estimates of cyanobacterial gene abundance beginning in ~1968 occurred in Lake 227, in keeping with experimental addition of nutrients and increase in planktonic cyanobacteria. In contrast, no significant rise in cyanobacterial abundance associated with lake fertilization was observed with HTS. Relative abundances of Microcystis between the two techniques showed moderate to strong levels of coherence in top intervals of the sediment cores. Both ddPCR and HTS approaches are suitable for sedDNA analysis, but studies aiming to quantify absolute abundances from complex environments should consider using ddPCR due to its high tolerance to PCR inhibitors.

Water pre-filtration methods to improve environmental DNA detection by real-time PCR and metabarcoding

Environmental DNA (eDNA) analysis is a novel approach for biomonitoring and has been mostly used in clear water. It is difficult to detect eDNA in turbid water as filter clogging occurs, and environmental samples contain various substances that inhibit the polymerase chain reaction (PCR) and affect the accuracy of eDNA analysis. Therefore, we applied a pre-filtration method to better detect the fish species (particularly pale chub, Opsariichthys platypus) present in a water body by measuring eDNA in environmental samples containing PCR inhibitors. Upon conducting 12S rRNA metabarcoding analysis (MiFish), we found that pre-filtration did not affect the number or identities of fish species detected in our samples, but pre-filtration through pore sizes resulted in significantly reduced variance among replicate samples. Additionally, PCR amplification was improved by the pre-filtration of environmental samples containing PCR inhibitors such as humic substances. Although this study may appear to be a conservative and ancillary experiment, pre-filtration is a simple technique that can not only improve the physical properties of water, such as turbidity, but also the quality of eDNA biomonitoring.

Evaluation of an Automated High-Throughput Liquid-Based RNA Extraction Platform on Pooled Nasopharyngeal or Saliva Specimens for SARS-CoV-2 RT-PCR

SARS-CoV-2 RT-PCR with pooled specimens has been implemented during the COVID-19 pandemic as a cost- and manpower-saving strategy for large-scale testing. However, there is a paucity of data on the efficiency of different nucleic acid extraction platforms on pooled specimens. This study compared a novel automated high-throughput liquid-based RNA extraction (LRE) platform (PHASIFYTM) with a widely used magnetic bead-based total nucleic acid extraction (MBTE) platform (NucliSENS® easyMAG®). A total of 60 pools of nasopharyngeal swab and 60 pools of posterior oropharyngeal saliva specimens, each consisting of 1 SARS-CoV-2 positive and 9 SARS-CoV-2 negative specimens, were included for the comparison. Real-time RT-PCR targeting the SARS-CoV-2 RdRp/Hel gene was performed, and GAPDH RT-PCR was used to detect RT-PCR inhibitors. No significant differences were observed in the Ct values and overall RT-PCR positive rates between LRE and MBTE platforms (92.5% (111/120] vs 90% (108/120]), but there was a slightly higher positive rate for LRE (88.3% (53/60]) than MBTE (81.7% (49/60]) among pooled saliva. The automated LRE method is comparable to a standard MBTE method for the detection of SAR-CoV-2 in pooled specimens, providing a suitable alternative automated extraction platform. Furthermore, LRE may be better suited for pooled saliva specimens due to more efficient removal of RT-PCR inhibitors.

Redesigning SARS-CoV-2 clinical RT-qPCR assays for wastewater RT-ddPCR

COVID-19 wastewater surveillance has gained widespread acceptance to monitor community infection trends. Wastewater samples primarily differ from clinical samples by having low viral concentrations due to dilution, and high levels of PCR inhibitors. Therefore, wastewater samples should have appropriately designed and optimized molecular assays. Digital PCR has proven to be more sensitive and resilient to matrix inhibition. However, most SARS-CoV-2 assays being used have been designed for clinical use on RT-qPCR instruments, then adopted to digital PCR platforms. But it is unknown whether clinical RT-qPCR assays are adequate to use on digital PCR platforms. Here we designed an N- and E- gene multiplex (ddCoV_N and ddCoV_E) specifically for RT-ddPCR and benchmarked them against the nCoV_N2 and E_Sarbeco assays. ddCoV_N and ddCoV_E have equivalent limits of detections and samples concentrations to NCoV_N2 and E_Sarbeco but showed improved signal-to-noise ratios that eased interpretation and ability to multiplex. From GISAID downloaded unique sequences analyzed, 2.12% and 0.83% present a mismatch or would not be detected by the used primer/probe combination for the ddCoV_N and ddCoV_E, respectively.

Methods to improve molecular detection of Salmonella in complex herbal matrices containing inhibitors

Salmonella is one of the main causes of foodborne diseases worldwide. Molecular tests such as the polymerase chain reaction (PCR) are rapid and sensitive and are increasingly becoming a preferred method for pathogen detection. However, the presence of PCR inhibiting substances in the analyzed samples could reduce the sensitivity or totally inhibit PCR amplification, which might result in failure of detection of the pathogen. Using multiplex real-time PCR, I investigated the detection of Salmonella enterica serovar Typhimurium in three herbal matrices containing inhibiting substances: i) chamomile (Matricaria recutita); ii) sage (Salvia officinalis) and iii) mint (Menthae piperitae). Internal positive controls (IPC) in the multiplex PCR reactions indicated the degree of inhibition. All of the three herbs inhibited PCR amplification at standard concentration of the matrix (10% suspensions). I applied and compared four approaches to overcome the negative effect of the matrices on the PCR detection of Salmonella. The efficiency strongly depended on the matrix and the method used for removing the inhibiting substances. By a series of centrifugation steps combined with direct PCR, I managed to remove the PCR inhibitors and successfully detected the pathogen in each of the tested matrices. Moreover, this approach did not decrease significantly the PCR sensitivity and the detection of the pathogen was with Cq delay of only 1.48 ± 1.05 cycle, compared to the control. Hence, by the proposed method, PCR detection of Salmonella became possible in matrices with strong inhibitory effect on the PCR reaction. In summary, a simple, efficient, reliable, quick and cost-effective generic approach for removal of PCR inhibitors, and detection of foodborne bacterial pathogens in complex matrices containing PCR inhibitors, was proposed.

The Utilization of Linear Polylysine Coupled with Mechanic Forces to Extract Microbial DNA from Different Matrices

Molecular approaches are powerful tools that are used for medical or environmental diagnoses. However, the main limitations of such a tools are that they extract low levels of DNA and they do not remove the inhibitors of polymerase chain reaction (PCR). Although the use of polycation to complex and purify DNA has been described in the literature, elution often requires a high ionic strength or pH levels not compatible with molecular analyses. In this paper, we described a new process that is based on the complexation of DNA with linear polylysine, followed by capturing the complex by a cation exchange resin. The originality of the process consisted of using mechanic force to elute DNA from the complex. The extraction method showed several advantages when compared to existing methods, such as being compatible with pH levels that range from 5 to 11, as well as high levels of DNA recovery and elimination of PCR inhibitors from complex samples. This method was successfully applied to different types of samples, such as environmental samples, beverage samples, and medical samples. Furthermore, it was proven to be a good solution for removing PCR inhibitors and assuring good DNA recovery yield.

RPA-PCR couple: an approach to expedite plant diagnostics and overcome PCR inhibitors

DNA extraction can be lengthy and sometimes ends up with amplification inhibitors. We present the potential of recombinase polymerase amplification (RPA) to replace plant DNA extraction. In our rapid ‘RPA-PCR couple’ concept, RPA is tuned to slower reaction kinetics to promote amplification of long targets. RPA primers amplify target and some flanking regions directly from simple plant macerates. Then PCR primers exponentially amplify the target directly from the RPA reaction. We present the coupling of RPA with conventional, TaqMan and SYBR Green PCR assays. We applied the concept to strawberry Phytophthora pathogens and the Phytophthora identification marker atp9-nad9. We found RPA-PCR couple specific, sensitive and reliable. The approach may also benefit other difficult samples such as food, feces and ancient samples.