Development and evaluation of a ligation-free sequence-independent, single-primer amplification (LF-SISPA) assay for whole genome characterization of viruses.

Molecular identification and characterization of novel or re-emerging infectious pathogens is critical for disease surveillance and outbreak investigations. Next generation sequencing (NGS) using Sequence-Independent, Single-Primer Amplification (SISPA) is being used extensively in sequencing of viral genomes but it requires an expensive library preparation step. We developed a simple, low-cost method that enriches nucleic acids followed by a ligation-free (LF) 2-step Polymerase Chain Reaction (PCR) procedure for library preparation. A pan-chimeric universal primer (JS15N14) containing 15 nucleotides with a random tetradecamer (14N) attached to the 3'-end was designed. The complimentary primer (JS15) was used for nucleic acid enrichment in a first round PCR. A second PCR was designed to create Illumina sequencer-compatible sequencing-ready libraries for NGS. The new LF-SISPA protocol was tested using six RNA and DNA viral genomes (10.8-229.4 kilobases, kb) from an ATCC virome nucleic acid mix (ATCC® MSA-1008™) followed by analysis using One Codex, an online identification tool. In addition, a human stool sample known to be positive for norovirus GII was sequenced, and de novo assembly was performed using the Genome Detective Virus Tool allowing for near complete genome identification in less than 24 h. The LF-SISPA method does not require prior knowledge of target sequences and does not require an expensive enzymatic library preparation kit, thereby providing a simple, fast, low-cost alternative for the identification of unknown viral pathogens.

Removal and Inactivation of an Enveloped Virus Surrogate by Iron Conventional Coagulation and Electrocoagulation.

It is imperative to understand the behavior of enveloped viruses during water treatment to better protect public health, especially in the light of evidence of detection of coronaviruses in wastewater. We report bench-scale experiments evaluating the extent and mechanisms of removal and/or inactivation of a coronavirus surrogate (ϕ6 bacteriophage) in water by conventional FeCl3 coagulation and Fe(0) electrocoagulation. Both coagulation methods achieved ∼5-log removal/inactivation of ϕ6 in 20 min. Enhanced removal was attributed to the high hydrophobicity of ϕ6 imparted by its characteristic phospholipid envelope. ϕ6 adhesion to freshly precipitated iron (hydr)oxide also led to envelope damage causing inactivation in both coagulation techniques. Fourier transform infrared spectroscopy revealed oxidative damages to ϕ6 lipids only for electrocoagulation consistent with electro-Fenton reactions. Monitoring ϕ6 dsRNA by a novel reverse transcription quantitative polymerase chain reaction (RT-qPCR) method quantified significantly lower viral removal/inactivation in water compared with the plaque assay demonstrating that relying solely on RT-qPCR assays may overstate human health risks arising from viruses. Transmission electron microscopy and computationally generated electron density maps of ϕ6 showed severe morphological damages to virus' envelope and loss of capsid volume accompanying coagulation. Both conventional and electro- coagulation appear to be highly effective in controlling enveloped viruses during surface water treatment.

A new solid matrix for preservation of viral nucleic acid from clinical specimens at ambient temperature.

Stabilizing paper matrix methods for retaining nucleic acid from inactivated clinical specimens offer a solution for molecular diagnostics when specimens may be stored or shipped at ambient temperature. We developed cellulose disks (UNEXP) saturated with a total nucleic acid extraction buffer (UNEX) modified from a previously developed lysis buffer for multiple enteric pathogens. Infectivity of hepatitis A virus, adenovirus and poliovirus was destroyed after 2-3 h incubation at room temperature on the UNEXP disks. Norovirus RNA could be detected in UNEXP-eluted nucleic acids by reverse transcription-quantitative PCR (RT-qPCR) from 54 stool samples after 2 weeks storage at room temperature on disks; a subset of seven samples were positive after 3 months storage. Genotyping was successful in 76% of 54 samples tested including six of seven samples stored on the UNEXP disks for up to one month. Comparison of UNEXP with the FTA elute card in a subset of 10 samples demonstrated similar detection and genotyping rates after two weeks of storage at room temperature. UNEXP disks could be useful for epidemiologic investigations of disease outbreaks in resource-limited areas by simplifying specimen transport to regional diagnostic laboratories or shipment to international centers without the need to ship samples on dry ice.

Pool water quality and prevalence of microbes in filter backwash from metro-Atlanta swimming pools.

During the 2012 summer swim season, aquatic venue data and filter backwash samples were collected from 127 metro-Atlanta pools. Last-recorded water chemistry measures indicated 98% (157/161) of samples were from pools with ≥1 mg/L residual chlorine without stabilized chlorine or ≥2 mg/L with stabilized chlorine and 89% (144/161) had pH readings 7.2-7.8. These water quality parameters are consistent with the 2016 Model Aquatic Health Code (2nd edition) recommendations. We used previously validated real-time polymerase chain reaction assays for detection of seven enteric microbes, including Escherichia coli, and Pseudomonas aeruginosa. E. coli was detected in 58% (93/161) of samples, signifying that swimmers likely introduced fecal material into pool water. P. aeruginosa was detected in 59% (95/161) of samples, indicating contamination from swimmers or biofilm growth on surfaces. Cryptosporidium spp. and Giardia duodenalis were each detected in approximately 1% of samples. These findings indicate the need for aquatics staff, state and local environmental health practitioners, and swimmers to each take steps to minimize the risk of transmission of infectious pathogens.

Real-time PCR and sequencing assays for rapid detection and identification of avian schistosomes in environmental samples.

Cercarial dermatitis, also known as swimmer's itch, is an allergenic skin reaction followed by intense itching caused by schistosome cercariae penetrating human skin. Cercarial dermatitis outbreaks occur globally and are frequently associated with freshwater lakes and are occasionally associated with marine or estuarine waters where birds reside year-round or where migratory birds reside. In this study, a broadly reactive TaqMan assay targeting 18S rRNA gene (ribosomal DNA [rDNA]) sequences that was based on a genetically diverse panel of schistosome isolates representing 13 genera and 20 species (the 18S rDNA TaqMan assay) was developed. A PCR assay was also developed to amplify a 28S rDNA region for subsequent sequencing to identify schistosomes. When applied to surface water samples seeded with Schistosoma mansoni cercariae, the 18S rDNA TaqMan assay enabled detection at a level of 5 S. mansoni cercariae in 100 liters of lake water. The 18S rDNA TaqMan and 28S rDNA PCR sequencing assays were also applied to 100-liter water samples collected from lakes in Nebraska and Wisconsin where there were reported dermatitis outbreaks. Avian schistosome DNA was detected in 11 of 34 lake water samples using the TaqMan assay. Further 28S rDNA sequence analysis of positive samples confirmed the presence of avian schistosome DNA and provided a preliminary identification of the avian schistosomes in 10 of the 11 samples. These data indicate that the broadly schistosome-reactive TaqMan assay can be effective for rapid screening of large-volume water samples for detection of avian schistosomes, thereby facilitating timely response actions to mitigate or prevent dermatitis outbreaks. Additionally, samples positive by the 18S rDNA TaqMan assay can be further assayed using the 28S rDNA sequencing assay to both confirm the presence of schistosomes and contribute to their identification.

Draft Genome Sequence of Buttiauxella agrestis, Isolated from Surface Water.

MI agar is routinely used for quantifying Escherichia coli in drinking water. A suspect E. coli colony isolated from a water sample was identified as Buttiauxella agrestis. The whole genome sequence of B. agrestis was determined to understand the genetic basis for its phenotypic resemblance to E. coli on MI agar.

Draft Genome Sequence of Raoultella planticola, Isolated from River Water.

We isolated Raoultella planticola from a river water sample, which was phenotypically indistinguishable from Escherichia coli on MI agar. The genome sequence of R. planticola was determined to gain information about its metabolic functions contributing to its false positive appearance of E. coli on MI agar. We report the first whole genome sequence of Raoultella planticola.

Rapid detection of microbial DNA by a novel isothermal genome exponential amplification reaction (GEAR) assay.

In this study we report the development of a simple target-specific isothermal nucleic acid amplification technique, termed genome exponential amplification reaction (GEAR). Escherichia coli was selected as the microbial target to demonstrate the GEAR technique as a proof of concept. The GEAR technique uses a set of four primers; in the present study these primers targeted 5 regions on the 16S rRNA gene of E. coli. The outer forward and reverse Tab primer sequences are complementary to each other at their 5' end, whereas their 3' end sequences are complementary to their respective target nucleic acid sequences. The GEAR assay was performed at a constant temperature 60 °C and monitored continuously in a real-time PCR instrument in the presence of an intercalating dye (SYTO 9). The GEAR assay enabled amplification of as few as one colony forming units of E. coli per reaction within 30 min. We also evaluated the GEAR assay for rapid identification of bacterial colonies cultured on agar media directly in the reaction without DNA extraction. Cells from E. coli colonies were picked and added directly to GEAR assay mastermix without prior DNA extraction. DNA in the cells could be amplified, yielding positive results within 15 min.

Hollow-fiber ultrafiltration for simultaneous recovery of viruses, bacteria and parasites from reclaimed water.

Hollow-fiber ultrafiltration (UF) is a technique that has been reported to be effective for recovering a diverse array of microbes from water, and may also be potentially useful for microbial monitoring of effluent from water reclamation facilities. However, few data are available to indicate the potential limitations and efficacy of the UF technique for treated wastewater. In this study, recovery efficiencies were determined for various options available for performing the tangential-flow UF technique, including hollow-fiber ultrafilter (i.e., dialyzer) type, ultrafilter pre-treatment (i.e., blocking), and elution. MS2 and ΦX174 bacteriophages, Clostridium perfringens spores, Escherichia coli, and Cryptosporidium parvum oocysts were seeded into 10-L reclaimed water samples to evaluate UF options. Then a single UF protocol was established and studied using seeded and non-seeded 100-L samples from two water reclamation facilities in Georgia, USA. Baxter Exeltra Plus 210 and Fresenius F200NR dialyzers were found to provide significantly higher microbial recovery than Minntech HPH 1400 hemoconcentrators. The selected final UF method incorporated use of a non-blocked ultrafilter for UF followed by elution using a surfactant-based solution. For 10-L samples, this method achieved recovery efficiencies of greater than 50% recovery of seeded viruses, bacteria, and parasites. There was no significant difference in overall microbial recovery efficiency when the method was applied to 10- and 100-L samples. In addition, detection levels for pathogens in seeded 100-L reclaimed water samples were 1000 PFU HAV, 10,000 GI norovirus particles, <500 Salmonella and <200 Cryptosporidium oocysts. These data demonstrate that UF can be an effective technique for recovering diverse microbes in reclaimed water to monitor and improve effluent water quality in wastewater treatment plants.

Evaluation of a molecular beacon real-time PCR assay for detection of Baylisascaris procyonis in different soil types and water samples.

Baylisascaris procyonis is a helminth parasite commonly found in North American raccoons (Procyon lotor) that is a cause of clinical neural, ocular, and visceral larva migrans in humans when infective eggs are ingested. Rapid detection of B. procyonis eggs in contaminated soil and water would assist public health analysts in evaluating risks associated with public exposure to areas of known raccoon activity. In this study, a molecular beacon probe-based real-time polymerase chain reaction (PCR) assay was developed to enable rapid and specific detection of eggs of Baylisascaris spp. The molecular beacon assay targeted the cytochrome oxidase subunit 2 (cox-2) gene of B. procyonis. To determine method sensitivity, experiments testing various egg levels (250, 25, and five eggs) were performed by seeding into 0.5-g soil samples or 0.5-mL water samples. Different soil sample types were extracted using a commercial nucleic acid extraction kit. Specificity testing using previously characterized helminth tissue specimens indicated that the assay was specific to Baylisascaris spp. Little real-time PCR inhibition was observed for most of the soil and water samples. A seed level of 250 eggs was detected for all soil types, and two seed levels (25 and five eggs) were detected for surface water samples. These results demonstrate that the reported real-time PCR assay was effective for the sensitive detection of B. procyonis in a wide range of soil types, and should be a useful tool for investigations of soil or water potentially contaminated with eggs of this parasite.

Multistate evaluation of an ultrafiltration-based procedure for simultaneous recovery of enteric microbes in 100-liter tap water samples.

Ultrafiltration (UF) is increasingly being recognized as a potentially effective procedure for concentrating and recovering microbes from large volumes of water and treated wastewater. Because of their very small pore sizes, UF membranes are capable of simultaneously concentrating viruses, bacteria, and parasites based on size exclusion. In this study, a UF-based water sampling procedure was used to simultaneously recover representatives of these three microbial classes seeded into 100-liter samples of tap water collected from eight cities covering six hydrologic areas of the United States. The UF-based procedure included hollow-fiber UF as the primary step for concentrating microbes and then used membrane filtration for bacterial culture assays, immunomagnetic separation for parasite recovery and quantification, and centrifugal UF for secondary concentration of viruses. Water samples were tested for nine water quality parameters to investigate whether water quality data correlated with measured recovery efficiencies and molecular detection levels. Average total method recovery efficiencies were 71, 97, 120, 110, and 91% for phiX174 bacteriophage, MS2 bacteriophage, Enterococcus faecalis, Clostridium perfringens spores, and Cryptosporidium parvum oocysts, respectively. Real-time PCR and reverse transcription-PCR (RT-PCR) for seeded microbes and controls indicated that tap water quality could affect the analytical performance of molecular amplification assays, although no specific water quality parameter was found to correlate with reduced PCR or RT-PCR performance.

A broadly reactive one-step real-time RT-PCR assay for rapid and sensitive detection of hepatitis E virus.

Hepatitis E virus (HEV) is transmitted by the fecal-oral route and causes sporadic and epidemic forms of acute hepatitis. Large waterborne HEV epidemics have been documented exclusively in developing countries. At least four major genotypes of HEV have been reported worldwide: genotype 1 (found primarily in Asian countries), genotype 2 (isolated from a single outbreak in Mexico), genotype 3 (identified in swine and humans in the United States and many other countries), and genotype 4 (identified in humans, swine and other animals in Asia). To better detect and quantitate different HEV strains that may be present in clinical and environmental samples, we developed a rapid and sensitive real-time RT-PCR assay for the detection of HEV RNA. Primers and probes for the real-time RT-PCR were selected based on the multiple sequence alignments of 27 sequences of the ORF3 region. Thirteen HEV isolates representing genotypes 1-4 were used to standardize the real-time RT-PCR assay. The TaqMan assay detected as few as four genome equivalent (GE) copies of HEV plasmid DNA and detected as low as 0.12 50% pig infectious dose (PID50) of swine HEV. Different concentrations of swine HEV (120-1.2PID50) spiked into a surface water concentrate were detected in the real-time RT-PCR assay. This is the first reporting of a broadly reactive TaqMan RT-PCR assay for the detection of HEV in clinical and environmental samples.

Quantitative real-time PCR assays for detection of human adenoviruses and identification of serotypes 40 and 41.

A quantitative real-time TaqMan PCR assay for detection of human adenoviruses (HAdV) was developed using broadly reactive consensus primers and a TaqMan probe targeting a conserved region of the hexon gene. The TaqMan assay correctly identified 56 representative adenovirus prototype strains and field isolates from all six adenovirus species (A to F). Based on infectious units, the TaqMan assay was able to detect as few as 0.4 and 0.004 infectious units of adenovirus serotype 2 (AdV2) and AdV41, respectively, with results obtained in less than 90 min. Using genomic equivalents, the broadly reactive TaqMan assay was able to detect 5 copies of AdV40 (which had zero mismatches with the PCR primers and probe), 8 copies of AdV41, and 350 copies of AdV3 (which had the most mismatches [seven] of any adenovirus serotype tested). For specific detection and identification of F species serotypes AdV40 and AdV41, a second real-time PCR assay was developed using fluorescence resonance energy transfer (FRET) probes that target the adenovirus fiber gene. The FRET-based assay had a detection limit of 3 to 5 copies of AdV40 and AdV41 standard DNA and was able to distinguish between AdV40 and AdV41 based on melting curve analysis. Both the TaqMan and FRET PCR assays were quantitative over a wide range of virus titers. Application of these assays for detection of adenoviruses and type-specific identification of AdV40 and AdV41 will be useful for identifying these viruses in environmental and clinical samples.

Rapid detection of infectious adenoviruses by mRNA real-time RT-PCR.

Adenoviruses are among the most persistent and ubiquitous viruses in water and wastewater, but some of them are difficult to detect due to non-cytopathogenicity and slow growth in cell cultures. A TaqMan real-time RT-PCR method in conjunction with cell culture infectivity was developed to rapidly detect mRNA produced by infectious adenoviruses in water samples. Only infectious adenoviruses were detected by this method, based on their ability to produce mRNA during replication in cell culture. The mRNA of Ad41 was detected as soon as 3 days after infection at levels as low as 5 infectious units (IU) per cell culture. In order to confirm that our methods detected only infectious viruses, 1-ml volumes of 10(4)IU of Ad41 were exposed to different free chlorine doses. No mRNA was detected in cells inoculated with Ad41 treated with the highest free chlorine dose of 100 mg min/l. However, mRNA of adenovirus was detected in cells inoculated with virus that was untreated or exposed to a lower free chlorine dose of 1 mg min/l. These results suggest that mRNA detection by real-time RT-PCR is a sensitive and specific method to detect low levels of infectious adenoviruses in water and other environmental media within 1-3 days.

Rapid and sensitive detection of noroviruses by using TaqMan-based one-step reverse transcription-PCR assays and application to naturally contaminated shellfish samples.

Noroviruses (NoV), which are members of the family Caliciviridae, are the most important cause of outbreaks of acute gastroenteritis worldwide and are commonly found in shellfish grown in polluted waters. In the present study, we developed broadly reactive one-step TaqMan reverse transcription (RT)-PCR assays for the detection of genogroup I (GI) and GII NoV in fecal samples, as well as shellfish samples. The specificity and sensitivity of all steps of the assays were systematically evaluated, and in the final format, the monoplex assays were validated by using RNA extracted from a panel of 84 stool specimens, which included NoV strains representing 19 different genotypes (7 GI, 11 GII, and 1 GIV strains). The assays were further validated with 38 shellfish cDNA extracts previously tested by nested PCR. Comparison with a recently described real-time assay showed that our assay had significantly higher sensitivity and was at least as sensitive as the nested PCR. For stool specimens, a one-step duplex TaqMan RT-PCR assay performed as well as individual genogroup-specific monoplex assays. All other enteric viruses examined were negative, and no cross-reaction between genogroups was observed. These TaqMan RT-PCR assays provide rapid (less than 90 min), sensitive, and reliable detection of NoV and should prove to be useful for routine monitoring of both clinical and shellfish samples.

Enrichment and DNA extraction protocols for the simultaneous detection of Salmonella and Listeria monocytogenes in raw sausage meat with multiplex real-time PCR.

A novel method of DNA extraction and purification was developed and was used in conjunction with a multiplex real-time PCR assay for the simultaneous detection of Salmonella and Listeria monocytogenes in a raw meat sample. The PCR used primers targeting the invA gene of Salmonella and the hlyA gene of L. monocytogenes, and PCR products were detected with a LightCycler on the basis of fluorescence from SYBR Green and melting temperature. The assay allowed the detection of 3 Listeria cells and 4 Salmonella cells per g of the original sausage within 10 h, including an enrichment period of 6 to 8 h.

Real-time multiplex SYBR green I-based PCR assay for simultaneous detection of Salmonella serovars and Listeria monocytogenes.

A multiplex SYBR Green I-based PCR assay has been developed for simultaneous detection of Salmonella serovars and Listeria monocytogenes using a LightCycler. Primers were designed to amplify an 85-bp sequence from the gene encoding a fimbrinlike protein (fimI) of Salmonella Enteritidis and a 98-bp sequence from the hemolysin gene (hly) of L. monocytogenes. These primers allowed the amplification of PCR products having distinct melting temperature values, resulting in the formation of two distinct peaks representing the two targets. Background signals, resulting from primer-dimer formation in the late cycles of PCR, are eliminated through the acquisition of data at a high temperature (>75 degrees C), but several degrees lower than required for detection of the specific PCR products. A rapid and simple method for the extraction of bacterial genomic DNA from liquid culture, coupled with duplex PCR using LightCycler SYBR Green-based PCR assays, detected the presence of 2.5 cells and 1 cell of Salmonella serovars and L. monocytogenes, respectively, within an hour. Following overnight enrichment, target DNA was present in sufficient quantities in 1 microl of culture to enable direct detection with the LightCycler.

Rapid detection of Escherichia coli O157:H7 with multiplex real-time PCR assays.

A SYBR Green LightCycler PCR assay using a single primer pair allowed simultaneous detection of stx1 and/or stx2 of Escherichia coli O157:H7. A distinct sequence of the Shiga-like toxin genes was amplified to yield products of 227 and/or 224 bp, respectively. The two products were distinguished by melting point curve analysis.