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.

Detection and identification of Giardia species using real-time PCR and sequencing.

We report a specific region of Giardia spp. 18S ribosomal RNA (18S rDNA) that serves as an ideal target for quantitative PCR (qPCR) detection and sequencing to identify Giardia species, including the clinically-relevant G. duodenalis, in clinical and environmental samples.

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 FeCl coagulation and Fe(0) electrocoagulation. Both coagulation methods achieved ∼5-log removal/inactivation of ϕ6 in 20 min.

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.

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.

Environmental Survey of Drinking Water Sources in Kampala, Uganda, during a Typhoid Fever Outbreak.

In 2015, a typhoid fever outbreak began in downtown Kampala, Uganda, and spread into adjacent districts. In response, an environmental survey of drinking water source types was conducted in areas of the city with high case numbers. A total of 122 samples was collected from 12 source types and tested for , free chlorine, and conductivity.

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.

Relative insignificance of virus inactivation during aluminum electrocoagulation of saline waters.

Combined removal and inactivation of the MS2 bacteriophage from model saline (0-100 mM NaCl) waters by electrochemical treatment using a sacrificial aluminum anode was evaluated. Both chemical and electrodissolution contributed to coagulant dosing since measured aluminum concentrations were statistically higher than purely electrochemical predictions using Faraday's law. Electrocoagulation generated only small amounts of free chlorine in situ but effectively destabilized viruses and incorporated them into Al(OH)3(s) flocs during electrolysis.

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.

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.

A novel photoinduced electron transfer (PET) primer technique for rapid real-time PCR detection of Cryptosporidium spp.

We report the development of a fluorescently labeled oligonucleotide primer that can be used to monitor real-time PCR. The primer has two parts, the 3'-end of the primer is complimentary to the target and a universal 17-mer stem loop at the 5'-end forms a hairpin structure. A fluorescent dye is attached to 5'-end of either the forward or reverse primer.

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.

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.

Development of an RNA extraction protocol for detection of waterborne viruses by reverse transcriptase quantitative PCR (RT-qPCR).

RNA extraction from environmental samples yields frequently an RNA preparation containing inhibitors of molecular reactions. Commercial RNA extraction kits commonly permit extraction of only 0.1-0.

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.

Broadly reactive TaqMan assay for real-time RT-PCR detection of rotavirus in clinical and environmental samples. JIN2@cdc.gov.

Rotaviruses are enteric pathogens responsible for a significant burden of disease, especially in children, through person-to-person transmission and exposure to contaminated food and water. In the present study, a TaqMan probe-based real-time reverse transcriptase (RT) polymerase chain reaction (PCR) assay was developed and validated for sensitive and specific detection and quantification of rotavirus for the routine screening of clinical and environmental samples. The assay primers and probes were designed to target the non-structural protein region 3 (NSP3) of rotavirus.

Detection and differentiation of Cryptosporidium hominis and Cryptosporidium parvum by dual TaqMan assays.

Rapid identification of the two major species of Cryptosporidium associated with human infections, Cryptosporidium hominis and Cryptosporidium parvum, is important for investigating outbreaks of cryptosporidiosis. This study reports the development and validation of a real-time PCR TaqMan procedure for detection of Cryptosporidium species and identification of C. hominis and C.

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.

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).

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.

Development and evaluation of a broadly reactive TaqMan assay for rapid detection of hepatitis A virus.

Primers and a TaqMan probe for the 5'-untranslated region (UTR) of the hepatitis A virus (HAV) genome were designed and evaluated. The assay detected 0.5 infectious units of HAV and 40 copies of a synthetic transcript and provides an important screening tool for rapid quantitative HAV detection in clinical or 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.

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).

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.