Last data update: Jan 27, 2025. (Total: 48650 publications since 2009)
Records 1-18 (of 18 Records) |
Query Trace: Jothikumar N[original query] |
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Development and evaluation of a ligation-free sequence-independent, single-primer amplification (LF-SISPA) assay for whole genome characterization of viruses
Jothikumar N , Cromeans T , Shivajothi J , Vinjé J , Murphy J . J Virol Methods 2021 299 114346 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.
Kim K , Jothikumar N , Sen A , Murphy JL , Chellam S . Environ Sci Technol 2021 55 (4) 2674-2683 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(3) 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.
Cromeans T , Jothikumar N , Lee J , Collins N , Burns CC , Hill VR , Vinje J . J Virol Methods 2019 274 113732 ![]() ![]() 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
Murphy JL , Hlavsa MC , Carter BC , Miller C , Jothikumar N , Gerth TR , Beach MJ , Hill VR . J Water Health 2018 16 (1) 87-92 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. |
Environmental survey of drinking water sources in Kampala, Uganda during a typhoid fever outbreak
Murphy JL , Kahler AM , Nansubuga I , Nanyunja EM , Kaplan B , Jothikumar N , Routh J , Gomez GA , Mintz ED , Hill VR . Appl Environ Microbiol 2017 83 (23) 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 were collected from 12 source types and tested for E. coli, free chlorine, and conductivity. An additional 37 grab samples from seven source types and 16 paired large volume (20-L) samples from wells and springs were also collected and tested for the presence of Salmonella Typhi. Escherichia coli was detected in 60% of kaveras (drinking water sold in plastic bags) and 80% of refilled water bottles; free chlorine was not detected in either source type. Most jerry cans (68%) contained E. coli and had free chlorine residuals below the WHO recommended level of 0.5 mg/L during outbreaks. Elevated conductivity readings for kaveras, refilled water bottles, and jerry cans (compared to treated surface water supplied by the water utility) suggested they likely contained untreated ground water. All unprotected springs and wells and more than 60% of protected springs contained E. coli Water samples collected from the water utility were found to have acceptable free chlorine levels and no detectable E. coli While S Typhi was not detected in water samples, Salmonella spp. were detected in samples from two unprotected springs, one protected spring, and one refilled water bottle. These data provided clear evidence that unregulated vended water and ground water represented a risk for typhoid transmission.Importance Despite the high incidence of typhoid fever globally, relatively few outbreak investigations incorporate drinking water testing. During waterborne disease outbreaks, measurement of physical-chemical parameters, such as free chlorine residual and electrical conductivity, and microbiological parameters, such as the presence of E. coli or the implicated etiologic agent, in drinking water samples can identify contaminated sources. This investigation indicated that unregulated vended water and ground water sources were contaminated and were therefore a risk to consumers during the 2015 typhoid fever outbreak in Kampala. Identification of contaminated drinking water sources and sources that do not contain adequate disinfectant levels can lead to rapid targeted interventions. |
Development of a nucleic Acid extraction procedure for simultaneous recovery of DNA and RNA from diverse microbes in water.
Hill VR , Narayanan J , Gallen RR , Ferdinand KL , Cromeans T , Vinje J . Pathogens 2015 4 (2) 335-54 ![]() Drinking and environmental water samples contain a diverse array of constituents that can interfere with molecular testing techniques, especially when large volumes of water are concentrated to the small volumes needed for effective molecular analysis. In this study, a suite of enteric viruses, bacteria, and protozoan parasites were seeded into concentrated source water and finished drinking water samples, in order to investigate the relative performance of nucleic acid extraction techniques for molecular testing. Real-time PCR and reverse transcription-PCR crossing threshold (CT) values were used as the metrics for evaluating relative performance. Experimental results were used to develop a guanidinium isothiocyanate-based lysis buffer (UNEX buffer) that enabled effective simultaneous extraction and recovery of DNA and RNA from the suite of study microbes. Procedures for bead beating, nucleic acid purification, and PCR facilitation were also developed and integrated in the protocol. The final lysis buffer and sample preparation procedure was found to be effective for a panel of drinking water and source water concentrates when compared to commercial nucleic acid extraction kits. The UNEX buffer-based extraction protocol enabled PCR detection of six study microbes, in 100 L finished water samples from four drinking water treatment facilities, within three CT values (i.e., within 90% difference) of the reagent-grade water control. The results from this study indicate that this newly formulated lysis buffer and sample preparation procedure can be useful for standardized molecular testing of drinking and environmental waters. |
Real-time PCR and Sequencing Assays for Rapid Detection and Identification of Avian Schistosomes in Environmental Samples.
Narayanan J , Mull BJ , Brant SV , Loker ES , Collinson J , Secor WE , Hill VR . Appl Environ Microbiol 2015 81 (12) 4207-15 ![]() ![]() 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 fresh water lakes and occasionally with marine or estuarine waters where year-round or migratory birds reside. In this study, a broadly reactive TaqMan assay was developed targeting 18S ribosomal RNA (rDNA) gene sequences based on a genetically diverse panel of schistosome isolates representing 13 genera and 20 species. A PCR assay was also developed to amplify a 28S ribosomal RNA (rDNA) gene region for subsequent sequencing to identify schistosomes. When applied to surface water samples seeded with Schistosoma mansoni cercariae, the 18S TaqMan assay enabled detection at a level of 5 S. mansoni cercariae in 100 L of lake water. The 18S TaqMan and 28S PCR-sequencing assays were also applied to 100-L 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 sequence analysis of positive samples confirmed the presence, and provided preliminary identification of avian schistosomes in ten 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 TaqMan assay can be further assayed using the 28S sequencing assay to both to confirm the presence of schistosomes and contribute to their identification. |
Draft Genome Sequence of Buttiauxella agrestis, Isolated from Surface Water.
Jothikumar N , Kahler A , Strockbine N , Gladney L , Hill VR . Genome Announc 2014 2 (5) ![]() 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.
Jothikumar N , Kahler A , Strockbine N , Gladney L , Hill VR . Genome Announc 2014 2 (5) ![]() 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. |
Visual endpoint detection of Escherichia coli O157:H7 using isothermal Genome Exponential Amplification Reaction (GEAR) assay and malachite green.
Jothikumar P , Narayanan J , Hill V . J Microbiol Methods 2014 98 122-7 ![]() Rapid and specific detection methods for bacterial agents in drinking water are important for disease prevention and responding to suspected contamination events. In this study, an isothermal Genome Exponential Amplification Reaction (GEAR) assay for Escherichia coli O157:H7 was designed specifically to recognize a 199-bp fragment of the lipopolysaccharide gene (rfbE) for rapid testing of water samples. The GEAR assay was found to be specific for E. coli O157:H7 using 10 isolates of E. coli O157:H7 and a panel of 86 bacterial controls. The GEAR assay was performed at a constant temperature of 65 degrees C using SYTO 9 intercalating dye. Detection limits were determined to be 20CFU for the GEAR assay. When SYTO 9 fluorescence was measured using a real-time PCR instrument, the assay had the same detection limit as when malachite green was added to the reaction mix and a characteristic blue color was visually observed in positive reactions. The study also found that 50 and 20CFU of E. coli O157:H7 seeded into 100-liter of tap water could be detected by the GEAR assays after the sample was concentrated by hollow-fiber ultrafiltration (HFUF) and approximately 10% of HFUF concentrate was cultured using trypticase soy broth-novobiocin. When applied to 19 surface water samples collected from Tennessee and Kentucky, the GEAR assay and a published real-time PCR assay both detected E. coli O157:H7 in two of the samples. The results of this study indicate that the GEAR assay can be sensitive for rapid detection of E. coli O157:H7 in water samples using fluorometric instruments and visual endpoint determination. |
A novel photoinduced electron transfer (PET) primer technique for rapid real-time PCR detection of Cryptosporidium spp.
Jothikumar N , Hill V . Biochem Biophys Res Commun 2013 436 (2) 134-9 ![]() 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. The presence of guanosine residues at the first and second position of the 3'dangling end effectively quenches the fluorescence due to the photo electron transfer (PET) mechanism. During the synthesis of nucleic acid, the hairpin structure is linearized and the fluorescence of the incorporated primer increases several-fold due to release of the fluorescently labeled tail and the absence of guanosine quenching. As amplicons are synthesized during nucleic acid amplification, the fluorescence increase in the reaction mixture can be measured with commercially available real-time PCR instruments. In addition, a melting procedure can be performed to denature the double-stranded amplicons, thereby generating fluorescence peaks that can differentiate primer dimers and other non-specific amplicons if formed during the reaction. We demonstrated the application of PET-PCR for the rapid detection and quantification of Cryptosporidium parvum DNA. Comparison with a previously published TaqMan(R) assay demonstrated that the two real-time PCR assays exhibited similar sensitivity for a dynamic range of detection of 6000 to 0.6 oocysts per reaction. PET PCR primers are simple to design and less-expensive than dual-labeled probe PCR methods, and should be of interest for use by laboratories operating in resource-limited environments. |
Molecular diagnosis of malaria by photo-induced electron transfer fluorogenic primers: PET-PCR.
Lucchi NW , Narayanan J , Karell MA , Xayavong M , Kariuki S , Dasilva AJ , Hill V , Udhayakumar V . PLoS One 2013 8 (2) e56677 ![]() There is a critical need for developing new malaria diagnostic tools that are sensitive, cost effective and capable of performing large scale diagnosis. The real-time PCR methods are particularly robust for large scale screening and they can be used in malaria control and elimination programs. We have designed novel self-quenching photo-induced electron transfer (PET) fluorogenic primers for the detection of P. falciparum and the Plasmodium genus by real-time PCR. A total of 119 samples consisting of different malaria species and mixed infections were used to test the utility of the novel PET-PCR primers in the diagnosis of clinical samples. The sensitivity and specificity were calculated using a nested PCR as the gold standard and the novel primer sets demonstrated 100% sensitivity and specificity. The limits of detection for P. falciparum was shown to be 3.2 parasites/microl using both Plasmodium genus and P. falciparum-specific primers and 5.8 parasites/microl for P. ovale, 3.5 parasites/microl for P. malariae and 5 parasites/microl for P. vivax using the genus specific primer set. Moreover, the reaction can be duplexed to detect both Plasmodium spp. and P. falciparum in a single reaction. The PET-PCR assay does not require internal probes or intercalating dyes which makes it convenient to use and less expensive than other real-time PCR diagnostic formats. Further validation of this technique in the field will help to assess its utility for large scale screening in malaria control and elimination programs. |
Real-time loop-mediated isothermal amplification (RealAmp) for the species-specific identification of Plasmodium vivax.
Patel JC , Oberstaller J , Xayavong M , Narayanan J , Debarry JD , Srinivasamoorthy G , Villegas L , Escalante AA , Dasilva A , Peterson DS , Barnwell JW , Kissinger JC , Udhayakumar V , Lucchi NW . PLoS One 2013 8 (1) e54986 ![]() Plasmodium vivax infections remain a major source of malaria-related morbidity and mortality. Early and accurate diagnosis is an integral component of effective malaria control programs. Conventional molecular diagnostic methods provide accurate results but are often resource-intensive, expensive, have a long turnaround time and are beyond the capacity of most malaria-endemic countries. Our laboratory has recently developed a new platform called RealAmp, which combines loop-mediated isothermal amplification (LAMP) with a portable tube scanner real-time isothermal instrument for the rapid detection of malaria parasites. Here we describe new primers for the detection of P. vivax using the RealAmp method. Three pairs of amplification primers required for this method were derived from a conserved DNA sequence unique to the P. vivax genome. The amplification was carried out at 64 degrees C using SYBR Green or SYTO-9 intercalating dyes for 90 minutes with the tube scanner set to collect fluorescence signals at 1-minute intervals. Clinical samples of P. vivax and other human-infecting malaria parasite species were used to determine the sensitivity and specificity of the primers by comparing with an 18S ribosomal RNA-based nested PCR as the gold standard. The new set of primers consistently detected laboratory-maintained isolates of P. vivax from different parts of the world. The primers detected P. vivax in the clinical samples with 94.59% sensitivity (95% CI: 87.48-98.26%) and 100% specificity (95% CI: 90.40-100%) compared to the gold standard nested-PCR method. The new primers also proved to be more sensitive than the published species-specific primers specifically developed for the LAMP method in detecting P. vivax. |
Rapid detection of microbial DNA by a novel isothermal genome exponential amplification reaction (GEAR) assay.
Prithiviraj J , Hill V , Jothikumar N . Biochem Biophys Res Commun 2012 420 (4) 738-42 ![]() 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 degrees 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 30min. 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 15min. |
Hollow-fiber ultrafiltration for simultaneous recovery of viruses, bacteria and parasites from reclaimed water
Liu P , Hill VR , Hahn D , Johnson TB , Pan Y , Jothikumar N , Moe CL . J Microbiol Methods 2012 88 (1) 155-61 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 PhiX174 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. |
Detection of GI and GII noroviruses in ground water using ultrafiltration and TaqMan real-time RT-PCR
Hill VR , Mull B , Jothikumar N , Ferdinand K , Vinje J . Food Environ Virol 2010 2 (4) 218-224 Noroviruses (NoVs) are a leading cause of epidemic and sporadic acute gastrointestinal illness globally. These viruses can potentially contaminate rural private wells and non-community drinking water systems, and cause waterborne disease outbreaks related to consumption of contaminated ground water. Detection of NoVs in water samples can be challenging because they are genetically and antigenically diverse, and noncultivable. In the present study, the detection limits of a novel broadly reactive GI assay and an existing GII NoV real-time TaqMan reverse transcriptase-polymerase chain reaction (RT-qPCR) assay in ground water concentrates was determined. Ground water samples (50 l) from two sources (Lawrenceville, GA and Gainesville, FL, USA) were seeded with electron microscopy-enumerated and RT-qPCR quantified NoV and concentrated using hollow-fiber ultrafiltration (UF) followed by either polyethylene glycol (PEG) precipitation or microconcentrators. Detection limits for GI NoV ranged from 1 x 10^4 (GA source) to 2 x 10^5 (FL source) virus particles in 50 l water samples (corresponding to 200-3,000 particles/l) and 5 x 10^4 (GA source) to 5 x 10^5 (FL source) virus particles (corresponding to 1,000-10,000 particles/l) for GII NoV. The reported UF method, sample processing procedures, and RT-qPCR assays should be effective tools for sensitive detection of NoVs in large-volume water samples. 2010 Springer Science+Business Media, LLC (outside the USA). |
Development of an RNA extraction protocol for detection of waterborne viruses by reverse transcriptase quantitative PCR (RT-qPCR)
Jothikumar N , Sobsey MD , Cromeans TL . J Virol Methods 2010 169 (1) 8-12 ![]() 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 - 0.2ml sample volume. An RNA extraction buffer (RNAX buffer) was formulated for the extraction of viral RNA from 4.0ml using a silica column based protocol. To evaluate the RNAX buffer based protocol, we used hepatitis A virus (HAV) and coxsackievirus B3 (CVB3) to monitor the RNA extraction efficiency from environmental samples. For evaluation of viral RNA recovery from water concentrates which were prepared from river and pond water by PEG concentration. Serial ten fold dilutions of two waterborne viruses were added to the water concentrates for detection for evaluation by quantitative detection. Quantitative recovery of HAV and CVB3 was determined by reverse transcriptase quantitative real-time PCR (RT-qPCR). The extracted RNA was compatible with RT-qPCR and sensitivity of detection of 0.8 PFU per reaction was found with RNAX buffer and the developed protocol. This level of sensitivity was obtained using viral RNA extracted from 4.0ml of an inoculated water sample concentrate. The RNAX buffer developed in this study could be applicable to the detection of other pathogens in water and food. |
Evaluation of a molecular beacon real-time PCR assay for detection of Baylisascaris procyonis in different soil types and water samples
Gatcombe RR , Jothikumar N , Dangoudoubiyam S , Kazacos KR , Hill VR . Parasitol Res 2009 106 (2) 499-504 ![]() 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. |
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