PCR Inhibitors Research Articles

Use of β-cyclodextrin and milk protein-coated activated charcoal for rapid detection of Listeria monocytogenes in leafy greens by PCR without pre-enrichment

Listeriosis outbreaks caused by contaminated leafy greens leaf have increased in recent years, which can pose a serious threat to public health. Thus, a rapid, precise, and effective detection method is urgently needed. We reported a novel sample pre-treatment that utilizes β-cyclodextrin and milk protein-coated activated charcoal to remove PCR inhibitors in leafy green matrices and facilitate the recovery of L. monocytogenes, which can be coupled with PCR subsequently. The primers targeting the iap gene of L. monocytogenes were used for testing the specificity with 141 bacterial strains. Artificially contaminated leafy greens (100–105 CFU/25 g) that include spinach, lettuce, Lactuca sativa L., cole, and cabbage were tested to determine the sensitivity of the developed method. Natural leafy greens were tested to evaluate the application of the developed method compared with the conventional culture method. The stability (shelf life) of the reagents used in the developed method was determined using reagents stored at −20 °C for 3, 6, and 12 months to evaluate the sensitivity and application of the method. As a result, the specificity of the developed method was 100% and the detection limit was 101 CFU/25 g. The total detection time without pre-enrichment was 4 h. The detection rate of L. monocytogenes (spinach 5%, lettuce 3%, cole 1%, Lactuca sativa L. 0%, and cabbage 0%) and the detection rate of viable bacteria (100%) were consistent in both diagnostic methods (the coincidence rate was 100%). Furthermore, the sensitivity and the application of the developed method were not influenced using the reagents stored for 3, 6, and 12 months. The reagents could be used normally after storing at −20 °C for at least 12 months, which proved that these reagents have good stability under freezing conditions. This method for the detection of L. monocytogenes was rapid, sensitive, and specific, and can potentially be used to detect and monitor the target pathogen in leafy greens and to reduce the potential risk of L. monocytogenes for public health.

Evaluation of viral load in patients with Ebola virus disease in Liberia: a retrospective observational study

SummaryBackgroundViral load in patients with Ebola virus disease affects case fatality rate and is an important parameter used for diagnostic cutoffs, stratification in randomised controlled trials, and epidemiological studies. However, viral load in Ebola virus disease is currently estimated using numerous different assays and protocols that were not developed or validated for this purpose. Here, our aim was to conduct a laboratory-based re-evaluation of the viral loads of a large cohort of Liberian patients with Ebola virus disease and analyse these data in the broader context of the west Africa epidemic.MethodsIn this retrospective observational study, whole blood samples from patients at the Eternal Love Winning Africa Ebola treatment unit (Monrovia, Liberia) were re-extracted with an optimised protocol and analysed by droplet digital PCR (ddPCR) using a novel semi-strand specific assay to measure viral load. To allow for more direct comparisons, the ddPCR viral loads were also back-calculated to cycle threshold (Ct) values. The new viral load data were then compared with the Ct values from the original diagnostic quantitative RT-PCR (qRT-PCR) testing to identify differing trends and discrepancies.FindingsBetween Aug 28 and Dec 18, 2014, 727 whole blood samples from 528 individuals were collected. 463 (64%) were first-draw samples and 409 (56%) were from patients positive for Ebola virus (EBOV), species Zaire ebolavirus. Of the 307 first-draw EBOV-positive samples, 127 (41%) were from survivors and 180 (59%) were from non-survivors; 155 (50%) were women, 145 (47%) were men, and seven (2%) were not recorded, and the mean age was 29·3 (SD 15·0) years for women and 31·8 (SD 14·8) years for men. Survivors had significantly lower mean viral loads at presentation than non-survivors in both the reanalysed dataset (5·61 [95% CI 5·34–5·87] vs 7·19 [6·99–7·38] log10 EBOV RNA copies per mL; p<0·0001) and diagnostic dataset (Ct value 28·72 [27·97–29·47] vs 26·26 [25·72–26·81]; p<0·0001). However, the prognostic capacity of viral load increased with the reanalysed dataset (odds ratio [OR] of death 8·06 [95% CI 4·81–13·53], p<0·0001 for viral loads above 6·71 log10 EBOV RNA copies per mL vs OR of death 2·02 [1·27–3·20], p=0·0028 for Ct values below 27·37). Diagnostic qRT-PCR significantly (p<0·0001) underestimated viral load in both survivors and non-survivors (difference in diagnostic Ct value minus laboratory Ct value of 1·79 [95% CI 1·16–2·43] for survivors and 5·15 [4·43–5·87] for non-survivors). Six samples that were reported negative by diagnostic testing were found to be positive upon reanalysis and had high viral loads.InterpretationInaccurate viral load estimation from diagnostic Ct values is probably multifactorial; however, unaddressed PCR inhibition from tissue damage in patients with fulminant Ebola virus disease could largely account for the discrepancies observed in our study. Testing protocols for Ebola virus disease require further standardisation and validation to produce accurate viral load estimates, minimise false negatives, and allow for reliable epidemiological investigation.

In vitro activity of letermovir against human cytomegalovirus isolates with different drug susceptibility phenotypes

Letermovir (LTV) is approved for the prophylaxis of human cytomegalovirus (HCMV) infection in adult seropositive recipients of an allogeneic hematopoietic stem cell transplant. Here, we report on the in vitro activity of LTV against a large panel of clinical HCMV isolates and recombinant viruses with different drug susceptibility phenotypes to currently-approved DNA polymerase inhibitors or maribavir. No pre-existing mutations conferring resistance to LTV were detected by Sanger sequencing in clinical HCMV isolates susceptible or resistant to DNA polymerases inhibitors. The susceptibility of LTV against the different recombinant HCMV mutants with amino acid substitutions in the UL97 kinase or in the UL54 DNA polymerase was similar to that of the wild type virus. LTV was also effective against recombinant HCMV harboring UL97 mutations conferring resistance to maribavir.

Carrier RNA is a key factor affecting fully integrated short tandem repeats profiling in challenging forensic samples models

BackgroundShort tandem repeats (STRs) are used today to provide discriminatory power for DNA fingerprinting. The present results showed that different factors may affect STR profiles in challenging samples including DNA quantity, DNA quality, PCR inhibitors and storage time. In the present study, blood stain samples were applied on two types of fabrics (black cotton and denim) to compare the efficiency of two different DNA-extraction methods (automated magnetic based beads method (EZ1), and manual organic method), with and without adding carrier RNA molecules, and to assess the quality and quantity of the extracted DNA and their capabilities for producing reportable STR-profiles in the presence of PCR inhibitors at two different storage times.ResultsCarrier RNA caused a dramatic increase in DNA recovery from black cotton or denim using EZ1 in contrast to organic method. EZ1 was found to be preferred than organic, especially when a time passed over, while organic method was preferred when samples are available in small quantities. In addition, using carrier RNA within the organic method steps showed no improvement in STR profiling. EZ1 with carrier RNA was preferred for bloodstained samples on fabrics with textile dyes (black dye or denim indigo), especially when stored for a long time.ConclusionsDenim was found to be more problematic than black cotton due to presence of challenging inhibitors (indigo dye). DNA concentration, storage time and types of fabrics are key factors for choosing the appropriate extraction method for reportable STR profile. Using EZ1 with carrier RNA gives less dropout profile than not using it, or when using organic method even in presence or absence of carrier RNA. Anyway, innovation of more sensitive, more robust analytical protocols could result in a better understanding of these inhibitory samples.

UV Irradiation of Vaccinia Virus-Infected Cells Impairs Cellular Functions, Introduces Lesions into the Viral Genome, and Uncovers Repair Capabilities for the Viral Replication Machinery.

Vaccinia virus (VV), the prototypic poxvirus, encodes a repertoire of proteins responsible for the metabolism of its large dsDNA genome. Previous work has furthered our understanding of how poxviruses replicate and recombine their genomes, but little is known about whether the poxvirus genome undergoes DNA repair. Our studies here are aimed at understanding how VV responds to exogenous DNA damage introduced by UV irradiation. Irradiation of cells prior to infection decreased protein synthesis and led to an ∼12-fold reduction in viral yield. On top of these cell-specific insults, irradiation of VV infections at 4 h postinfection (hpi) introduced both cyclobutene pyrimidine dimer (CPD) and 6,4-photoproduct (6,4-PP) lesions into the viral genome led to a nearly complete halt to further DNA synthesis and to a further reduction in viral yield (∼35-fold). DNA lesions persisted throughout infection and were indeed present in the genomes encapsidated into nascent virions. Depletion of several cellular proteins that mediate nucleotide excision repair (XP-A, -F, and -G) did not render viral infections hypersensitive to UV. We next investigated whether viral proteins were involved in combatting DNA damage. Infections performed with a virus lacking the A50 DNA ligase were moderately hypersensitive to UV irradiation (∼3-fold). More strikingly, when the DNA polymerase inhibitor cytosine arabinoside (araC) was added to wild-type infections at the time of UV irradiation (4 hpi), an even greater hypersensitivity to UV irradiation was seen (∼11-fold). Virions produced under the latter condition contained elevated levels of CPD adducts, strongly suggesting that the viral polymerase contributes to the repair of UV lesions introduced into the viral genome. IMPORTANCE Poxviruses remain of significant interest because of their continuing clinical relevance, their utility for the development of vaccines and oncolytic therapies, and their illustration of fundamental principles of viral replication and virus/cell interactions. These viruses are unique in that they replicate exclusively in the cytoplasm of infected mammalian cells, providing novel challenges for DNA viruses. How poxviruses replicate, recombine, and possibly repair their genomes is still only partially understood. Using UV irradiation as a form of exogenous DNA damage, we have examined how vaccinia virus metabolizes its genome following insult. We show that even UV irradiation of cells prior to infection diminishes viral yield, while UV irradiation during infection damages the genome, causes a halt in DNA accumulation, and reduces the viral yield more severely. Furthermore, we show that viral proteins, but not the cellular machinery, contribute to a partial repair of the viral genome following UV irradiation.

Foscarnet-Type Inorganic-Organic Hybrid Nanoparticles for Effective Antiviral Therapy.

[ZrO]2+[(FCN)0.4(OH)0.8]2- and Gd3+[FCN]3- inorganic-organic hybrid nanoparticles (IOH-NPs) are novel saline antiviral nanocarriers with foscarnet (FCN) as a drug anion. FCN as a pyrophosphate analogue serves as a prototype of a viral DNA polymerase inhibitor. FCN is used for the treatment of herpesvirus infections, including the drug-resistant cytomegalovirus (CMV) and herpes simplex viruses, HSV-1 and HSV-2. The novel [ZrO]2+[(FCN)0.4(OH)0.8]2- and Gd3+[FCN]3- IOH-NPs are characterized by aqueous synthesis, small size (20-30 nm), low material complexity, high biocompatibility, and high drug load (up to 44 wt % FCN per nanoparticle). The antiviral activity of the FCN-type IOH-NPs is probed for the human cytomegalovirus (HCMV). Moreover, the uptake of FCN-type IOH-NPs into vesicles, cytoplasm, and nuclei of nonphagocytic lung epithelial cells is evaluated. As a result, a promising antiviral activity of the FCN-type IOH-NPs that significantly outperforms freely dissolved FCN at the level of clinical formulations is observed, encouraging a future use of FCN-type IOH-NPs for the delivery of antivirals against respiratory viruses.

DNA profiling from human bone cells in the absence of decalcification and DNA extraction.

Bone cells are a suitable substrate for DNA analysis if required to identify the person from whom a sample was taken. Osteocytes, the most abundant cell type in bone, are embedded within mineralized bone matrix. To release DNA from osteocytes for subsequent analyses, either demineralization of the mineral matrix or an overnight incubation is routinely carried out. In this study, we report on a simplified and rapid approach to analyze preserved bone samples that omits this lengthy decalcification process. Nine tibial bone samples were processed to release matrix-free bone cells after fragmentation without the use of liquid nitrogen. Cell morphology was assessed by microscopy at 220× magnification following staining with Diamond™ Nucleic Acid Dye. Based on the presence of stained nuclei, samples were processed either using a DNA extraction process or by a semi-direct PCR process. The analysis of the quantity and quality of DNA isolated by both methods was carried out by real-time PCR and STR profiling to assess inhibition of PCR and DNA degradation. All samples resulted in informative STR profiles with minimal indication of inhibitors. These results demonstrate a potential approach of STR profiling from matrix-free bone cells within 8hours without decalcification and DNA extraction.

PCR optimization for the detection of bunchy top virus of abaca (Musa textilis Nee) in Eastern Visayas Philippines

The bunchy top virus in Eastern Visayas has serverly reduced abaca production. Early and accurate detection of plant viral pathogens is an essential and crucial component for disease management. At present, there are no standard PCR conditions in the Eastern Visayas region for detecting the bunchy top virus at an early stage using PCR. Thus, optimization for the detection was carried out to assist in disease management. Different annealing temperatures (57, 60 and 65oC), gel concentrations (1, 1.5 and 2%), and running conditions (80, 90 and 100 volts) were tested using My TaqTM DNA Polymerase (Bioline, USA). The annealing temperatures of 57oC and 60oC resulted in DNA amplification as indicated by the presence of bands but absence of bands at 65oC. The higher voltages of 90 and 100 volts resulted in smears and distorted DNA bands with 1% and 1.5% agarose; thus 2% agarose gel was used to resolve small DNA fragments (100bp to 3kb). Electrophoresis using 80 volts for 45min successfully separated the DNA bands. The amplification of the product with internal control primers indicated the absence of PCR inhibitors in the abaca-extracted DNA samples. This confirmed the negative PCR reaction as indicative of the absence of the virus. The optimized PCR conditions could be applied by students and researchers for the early detection of bunchy top virus in the National Abaca Research Center Germplasm collection and the region.

Serial measurement of M. tuberculosis in blood from critically-ill patients with HIV-associated tuberculosis.

SummaryBackgroundDespite being highly prevalent in hospitalised patients with severe HIV-associated tuberculosis (TB) and sepsis, little is known about the mycobacteriology of Mycobacterium tuberculosis bloodstream infection (MTBBSI). We developed methods to serially measure bacillary load in blood and used these to characterise MTBBSI response to anti-TB therapy (ATT) and relationship with mortality.MethodsWe established a microscopy method for direct visualisation of M. tuberculosis bacilli in blood using a novel lysis-concentration protocol and the fluorescent probe, 4-N,N-dimethylaminonaphthalimide-trehalose (DMN-Tre). We tested blood using GeneXpert® MTB/RIF-Ultra (Xpert-ultra) and Myco/F lytic culture after processing blood through lysis-wash steps to remove PCR inhibitors and anti-microbial drug carry-over. HIV-positive patients predicted to have MTBBSI gave blood samples 0, 4, 24, 48 and 72 h after ATT initiation. Bacillary loads were quantified using microscopy, Xpert-ultra cycle threshold, and culture time-to-positivity. Pharmacodynamics were modelled using these measures combined on an ordinal scale, including association with 12-week mortality.FindingsM. tuberculosis was detected in 27 of 28 recruited participants; 25 (89%) by blood Xpert-ultra, 22 (79%) by DMN-Tre microscopy, and 21 (75%) by Myco/F lytic blood culture. Eight (29%) participants died by 12-week follow-up. In a combined pharmacodynamic model, predicted probabilities of negative DMN-Tre microscopy, blood Xpert-ultra, or blood culture after 72 h treatment were 0·64, 0·27, and 0·94, respectively, in those who survived, compared with 0·23, 0·06, and 0·71 in those who died (posterior probability of slower clearance of MTBBSI in those that died >0·99). DMN-Tre microscopy of blood demonstrated heterogenous bacillary morphologies, including microcolonies and clumps. Bacillary cell-length varied significantly with ATT exposure (mean cell-length increase 0·13 log-µm/day; 95%CrI 0·10–0·16).InterpretationPharmacodynamics of MTBBSI treatment can be captured using DMN-Tre microscopy, blood Xpert-ultra and culture. This could facilitate interventional trials in severe HIV-associated TB.FundingWellcome Trust, NIH Fogarty International Center, South African MRC, NIHR(UK), National Research Foundation of South Africa.

Development of an Accurate and Sensitive Diagnostic System Based on Conventional PCR for Detection of African Swine Fever Virus in Food Waste

African swine fever virus (ASFV), a highly contagious virus, can cause diseases with high mortality rates in pigs, making it a pathogen of social and economic significance. ASFV has been reported to show potential long-term survival in living livestock, such as pigs, but also in leftover cooking meat and undercooked pork meat. Hence, it is possible that there could be direct reinfection or secondary infection through feed produced from household food waste and treatment facilities. Many polymerase chain reaction (PCR)-based molecular diagnostic techniques to detect ASFV in clinical swine samples have been reported. However, those with applicability for food waste samples, which contain relatively low viral copy numbers and may contain various unknown inhibitors of PCR, are still lacking. In this study, we developed a conventional PCR-based diagnostic system that can detect ASFV with high sensitivity from food waste sample types. The technique shows a 10–100 times higher limit of detection compared to that of previously reported methods based on conventional PCR and quantitative real-time PCR. It is also capable of amplifying a sequence that is approximately 751 nucleotides, which is advantageous for similarity analysis and genotyping. Moreover, a ASFV-modified positive material different from ASFV that could synthesize 1400 nucleotide amplicons was developed to identify false-positive cases and thus enhance diagnostic accuracy. The method developed herein may be applicable for future ASFV monitoring, identification, and genotyping in food waste samples.

Assessment of mitochondrial toxicity in newborns and infants with congenital cytomegalovirus infection treated with valganciclovir

BackgroundGanciclovir/valganciclovir is currently indicated during the first 6 months of life in symptomatic children with congenital cytomegalovirus (CMV) infection. However, this treatment may have the potential to induce mitochondrial toxicity...

Molecular and Serological Detection of Leptospira spp. in Italian Tortoises (Testudo spp.)

Leptospirosis is a worldwide zoonosis, characterised by a complex ecological cycle, including the environment, wild and domestic animals, and humans. It is also considered a water-borne infection. Although a solid body of data is available for more conventional hosts, limited information is available on neglected species such as reptiles, especially Testudinidae, both captive and free-ranging. Molecular and serological tests were performed to detect Leptospira spp. in Testudo spp. in Italy. Leptospiral DNA was detected using cloacal swabs collected from 116 individuals (54 free-ranging and 62 captive tortoises) by polymerase chain reaction (PCR). Unfortunately, of these, swabs from only 50 animals were eligible for analysis due to the presence of PCR inhibitors. A serological microagglutination test was performed on 121 Testudo spp. (7 free-ranging and 114 rescued tortoises). Sixteen individuals were tested using both assays, while the remainder were tested with one or the other. Ten out of 50 (20%) amplifiable cloacal swabs were positive for Leptospira DNA, the identity of which was confirmed by sequencing two amplicons. Two out of four repeatedly sampled animals only tested positive in the autumn. Seroprevalence was relatively low (0.8%, 1/121), at low titre (1:100). No positivity was recorded among free-ranging animals. Although these results do not provide a complete epidemiological snapshot of leptospirosis in chelonians, they suggest that tortoises are susceptible to pathogenic Leptospira. Further data are needed to determine the role of the captive environment as a possible predisposing factor and to assess the role of these reptiles in the ecology of leptospirosis.

Developmental Validation of a Rapidly Mutating Y-STR Panel Labeled by Six Fluoresceins for Forensic Research

The male-specific region of the human Y chromosome is a useful genetic marker for genealogical searching, male inheritance testing, and male DNA mixture deconvolution in forensic studies. However, the Y chromosomal short tandem repeats (Y-STRs) are difficult to distinguish among related males due to their low/medium mutation rate. In contrast, rapidly mutating (RM) Y-STRs exhibit unusually high mutation rates and possess great potential for differentiating male lineages. In this study, we developed a novel Y-STRs multiplex amplification assay of 32 RM Y-STRs by fragment analysis using six dye-labeled technologies (FAM, HEX, TAMRA, ROX, VIG, and SIZ). The development and the validation of the kit were carried out in accordance with the Scientific Working Group guidelines on DNA Analysis Methods. Identical allelic profiles of the 32 RM Y-STRs using a DNA 9948 sample as the positive control could be observed at different concentrations of PCR reagents. Further, the RM Y-STRs did not show cross-reactions with other common animal species, and the developed assay could tolerate interferences from common PCR inhibitors and mixed DNA samples. More importantly, the kit showed relatively high sensitivity and could detect trace DNA samples. Genetic distributions of 32 RM Y-STRs in the Guizhou Han population revealed that these RM Y-STRs showed relatively high genetic diversities. In conclusion, the RM Y-STR assay developed here showed good species specificity, high sensitivity, tolerance to inhibitors, and sample compatibility, which can be viewed as a highly efficient tool with high discrimination capacity for forensic male differentiation.

Selection and Optimization of High-Yielding DNA Isolation Protocol for Quantitative Analyses of Methanogenic Archaea.

Methanogenic archaea are a functionally important component of the intestinal microbiota of humans and animals, participating in the utilization of detrimental hydrogen produced during gut fermentation. Despite this, archaeal DNA has rarely been found in intestinal microbiome analyses, which prompts the need to optimize detecting procedures of these microorganisms, including the DNA isolation step. Three commercially available kits for DNA isolation and one extra purification kit that removes PCR inhibitors were evaluated on chicken droppings. In addition, different variants of mechanical lysis and a double elution were tested to ensure the maximum efficiency of DNA isolation from archaea as well as bacteria. A quantitative real-time PCR was used to monitor the optimization progress. As a result, the combination of the selected Genomic Mini AX Bacteria+ kit with a 2-min-long sonication by ultrasonic probe and enzymatic pretreatment gave excellent extraction efficiency rates for DNA of methanogenic archaea (an approximate 50-fold increase compared to the standard enzymatic lysis described by the producer) and, at the same time, provided optimal protection of DNA extracted from bacteria susceptible to enzymatic lysis. The presented results indicate that the optimized protocol allows for highly efficient extraction of total DNA, which is well-suited for quantitative microbial analyses by real-time PCR.

Development of a real-time PCR assay for detection and differentiation of Mycoplasma ovipneumoniae and a novel respiratory-associated Mycoplasma species in domestic sheep and goats.

A novel respiratory-associated Mycoplasma species (M. sp. nov.) of unknown clinical significance was recently identified that causes false positive results with multiple published PCR methods reported to specifically detect Mycoplasma ovipneumonaie, a well-known respiratory pathogen in small ruminants. This necessitates our objective to develop a real-time PCR (qPCR) assay for improved specificity and sensitivity, and more rapid detection and differentiation of M. ovipneumoniae and the M. sp. nov. in domestic sheep (DS) and domestic goat (DG) samples, as compared to a conventional PCR and sequencing (cPCR-seq) assay. Primers and probes were designed based on available M. ovipneumoniae 16S rRNA gene sequences in the GenBank database, and partial 16S rRNA gene sequences provided by the United States Department of Agriculture, Agricultural Research Service (USDA-ARS) for M. ovipneumoniae and M. sp. nov. USDA-ARS provided DS (n=153) and DG (n=194) nasal swab nucleic acid that previously tested positive for either M. ovipneumoniae (n=117) or M. sp. nov. (n=138), or negative for both targets (n=92) by cPCR-seq. A host 18S rRNA gene was included as an internal control to monitor for the failure of nucleic acid extraction and possible PCR inhibition. For samples positive by cPCR-seq, qPCR agreement was 88.0% (103/117; κ=0.81) and 89.9% (124/138; κ=0.84) for M. ovipneumoniae and M. sp. nov., respectively; 12 of 255 (4.7%) cPCR-seq positive samples were qPCR positive for both targets. Of samples negative by cPCR for both mycoplasmas, qPCR detected M. ovipneumoniae and M. sp. nov. in 6.5% (6/92) and 4.3% (4/92), respectively. Samples with discordant results between the cPCR and sequencing assay and the new qPCR were analyzed by target sequencing; successfully sequenced samples had identity matches that confirmed the qPCR result. The increased target specificity of this qPCR is predicted to increase testing accuracy as compared to other published assays.

Direct identification of fungi associated with indoor and outdoor ornamental plants by utilizing PCR assay

Direct identification of fungi associated with indoor and outdoor ornamental plants by utilizing PCR assay

Comparable Results of Helicobacter pylori Antibiotic Resistance Testing of Stools vs Gastric Biopsies Using Next-Generation Sequencing

Comparable Results of Helicobacter pylori Antibiotic Resistance Testing of Stools vs Gastric Biopsies Using Next-Generation Sequencing

Comparison of RT-qPCR and RT-dPCR Platforms for the Trace Detection of SARS-CoV-2 RNA in Wastewater.

We compared reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and RT digital PCR (RT-dPCR) platforms for the trace detection of SARS-CoV-2 RNA in low-prevalence COVID-19 locations in Queensland, Australia, using CDC N1 and CDC N2 assays. The assay limit of detection (ALOD), PCR inhibition rates, and performance characteristics of each assay, along with the positivity rates with the RT-qPCR and RT-dPCR platforms, were evaluated by seeding known concentrations of exogenous SARS-CoV-2 in wastewater. The ALODs using RT-dPCR were approximately 2–5 times lower than those using RT-qPCR. During sample processing, the endogenous (n = 96) and exogenous (n = 24) SARS-CoV-2 wastewater samples were separated, and RNA was extracted from both wastewater eluates and pellets (solids). The RT-dPCR platform demonstrated a detection rate significantly greater than that of RT-qPCR for the CDC N1 and CDC N2 assays in the eluate (N1, p = 0.0029; N2, p = 0.0003) and pellet (N1, p = 0.0015; N2, p = 0.0067) samples. The positivity results also indicated that for the analysis of SARS-CoV-2 RNA in wastewater, including the eluate and pellet samples may further increase the detection sensitivity using RT-dPCR.

A Review of Isothermal Amplification Methods and Food-Origin Inhibitors against Detecting Food-Borne Pathogens.

The isothermal amplification method, a molecular-based diagnostic technology, such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA), is widely used as an alternative to the time-consuming and labor-intensive culture-based detection method. However, food matrices or other compounds can inhibit molecular-based diagnostic technologies, causing reduced detection efficiencies, and false-negative results. These inhibitors originating from food are polysaccharides and polyphenolic compounds in berries, seafood, and vegetables. Additionally, magnesium ions needed for amplification reactions can also inhibit molecular-based diagnostics. The successful removal of inhibitors originating from food and molecular amplification reaction is therefore proposed to enhance the efficiency of molecular-based diagnostics and allow accurate detection of food-borne pathogens. Among molecular-based diagnostics, PCR inhibitors have been reported. Nevertheless, reports on the mechanism and removal of isothermal amplification method inhibitors are insufficient. Therefore, this review describes inhibitors originating from food and some compounds inhibiting the detection of food-borne pathogens during isothermal amplification.

Detection and Sequencing of Multiple Human Norovirus Genotypes from Imported Frozen Raspberries Linked to Outbreaks in the Province of Quebec, Canada, in 2017

Human noroviruses are among the main causes of acute gastroenteritis worldwide. Frozen raspberries have been linked to several norovirus food-related outbreaks. However, the extraction of norovirus RNA from frozen raspberries remains challenging. Recovery yields are low and PCR inhibitors limit the sensitivity of the detection methodologies. In 2017, 724 people from various regions of the Province of Quebec, Canada, were infected by noroviruses and the outbreak investigation pointed to frozen raspberries as a putative source. A new magnetic silica bead approach was used for the extraction of viruses from different outbreak samples. The RNA extracts were tested by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and five samples were confirmed positive for norovirus by RT-qPCR amplicon sequencing. A multiplex long-range two-step RT-PCR approach was developed to amplify norovirus ORF2 and ORF3 capsid genes from the positive frozen raspberry RNA extracts and other sequencing strategies were also explored. These capsid genes were sequenced by Next-Generation Sequencing. Phylogenetic analyses confirmed the presence of multiple genotypes (GI.3, GI.6, and GII.17) and intra-genotype variants in some of the frozen raspberry samples. Variants of genotype GI.3 and GI.6 had 100% homology with sequences from patient samples. Similar strains were also reported in previous outbreaks. Confirmation approaches based on sequencing the norovirus capsid genes using Next-Generation Sequencing can be applied at trace level contaminations and could be useful to assess risk and assist in source tracking.