Comparative evaluation of Taqman real-time PCR and semi-nested VP1 PCR for detection of enteroviruses in clinical specimens

ArticleinJournal of clinical virology: the official publication of the Pan American Society for Clinical Virology 49(1):73-4 · September 2010with35 Reads
DOI: 10.1016/j.jcv.2010.06.022 · Source: PubMed
Abstract
Molecular diagnostic tests to detect enterovirus in clinical specimens usually target highly conserved sites in the 5'-non-translated region, allowing detection of all members of the genus. The sequences in the 5'-NTR do not correlate with serotype, but PCR and sequencing of the VP1 region can be used for typing; this system has largely replaced traditional antigenic typing. To investigate the relative performance of two common enterovirus assays. We compared the relative sensitivities of Taqman real-time RT-PCR (rRT-PCR) and a VP1 semi-nested PCR (RT-snPCR) assay in which sequencing the VP1 amplicon also provides typing information. There was 89% concordance between the two methods among the 371 clinical specimens tested (74 positive in both assays and 257 negative in both assays). Twenty-seven rRT-PCR-negative/VP1-positive specimens were confirmed positive by sequencing; 13 specimens were rRT-PCR-positive/VP1-negative. The results suggest that either assay can produce satisfactory results, but that using both assays in parallel should provide the highest sensitivity for clinical diagnostic testing.
    • "[21] , Ancylostoma duodenale [22], Ascaris lumbricoides, astrovirus, Bacteroides fragilis [23], Campylobacter spp. [24] , Clostridium difficile, Cryptosporidium hominus/parvum [25], Cyclospora cayetanensis [26], Cystoisospora belli [27], Encephalitozoon intestinalis [28], Entamoeba histolytica, Enterocytozoon bieneusi [28], enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC)/Shigella, enteropathogenic E. coli (EPEC), enterotoxigenic E.coli (ETEC), pan-enterovirus [29], Giardia lamblia, Helicobacter pylori, Mycobacterium tuberculosis [30], Necator americanus [22], norovirus GI [31], norovirus GII, rotavirus, Salmonella spp., sapovirus, Shiga-toxin producing E. coli (STEC), Strongyloides stercoralis [32]), Trichuris trichiura and Vibrio cholerae [21]. Virulence genes were used to define the E. coli pathotypes as follows: aaiC and/or aatA for EAEC, ipaH for EIEC/Shigella, ST and/or LT for ETEC, eae with or without bfpA for EPEC, and stx1 and/or stx2 for STEC. "
    [Show abstract] [Hide abstract] ABSTRACT: Background: Oral polio vaccine (OPV) and rotavirus vaccine (RV) exhibit poorer performance in low-income settings compared to high-income settings. Prior studies have suggested an inhibitory effect of concurrent non-polio enterovirus (NPEV) infection, but the impact of other enteric infections has not been comprehensively evaluated. Methods: In urban Bangladesh, we tested stools for a broad range of enteric viruses, bacteria, parasites, and fungi by quantitative PCR from infants at weeks 6 and 10 of life, coincident with the first OPV and RV administration respectively, and examined the association between enteropathogen quantity and subsequent OPV serum neutralizing titers, serum rotavirus IgA, and rotavirus diarrhea. Results: Campylobacter and enterovirus (EV) quantity at the time of administration of the first dose of OPV was associated with lower OPV1-2 serum neutralizing titers, while enterovirus quantity was also associated with diminished rotavirus IgA (-0.08 change in -log titer per tenfold increase in quantity; P=0.037), failure to seroconvert (OR 0.78, 95% CI: 0.64-0.96; P=0.022), and breakthrough rotavirus diarrhea (OR 1.34, 95% CI: 1.05-1.71; P=0.020) after adjusting for potential confounders. These associations were not observed for Sabin strain poliovirus quantity. Conclusion: In this broad survey of enteropathogens and oral vaccine performance we find a particular association between EV carriage, particularly NPEV, and OPV immunogenicity and RV protection. Strategies to reduce EV infections may improve oral vaccine responses. ClinicalTrials.gov Identifier: NCT01375647.
    Full-text · Article · May 2016
    • "All samples were analyzed for extraction and inhibition efficiency as described above (see Experimental Section), using enteric viruses that resulted negative in q-PCR analysis on all environmental samples (i.e., Human enteroviruses, HAV). Poliovirus 1 was chosen as reference material and a q-PCR, previously developed by Oberste et al. (Human enterovirus: HE) [42], as method of detection. Ten µL of aliquot B containing genomic equivalents (see Experimental Section) were used to test extraction efficiency and the other 10 µL diluted 10-and 100-fold to evaluate the inhibition. "
    [Show abstract] [Hide abstract] ABSTRACT: The transmission of water-borne pathogens typically occurs by a faecal-oral route, through inhalation of aerosols, or by direct or indirect contact with contaminated water. Previous molecular-based studies have identified viral particles of zoonotic and human nature in surface waters. Contaminated water can lead to human health issues, and the development of rapid methods for the detection of pathogenic microorganisms is a valuable tool for the prevention of their spread. The aims of this work were to determine the presence and identity of representative human pathogenic enteric viruses in water samples from six European countries by quantitative polymerase chain reaction (q-PCR) and to develop two quantitative PCR methods for Adenovirus 41 and Mammalian Orthoreoviruses. A 2-year survey showed that Norovirus, Mammalian Orthoreovirus and Adenoviruses were the most frequently identified enteric viruses in the sampled surface waters. Although it was not possible to establish viability and infectivity of the viruses considered, the detectable presence of pathogenic viruses may represent a potential risk for human health. The methodology developed may aid in rapid detection of these pathogens for monitoring quality of surface waters.
    Full-text · Article · Apr 2016
    • "Synthetic GII NoV RNA standards were purchased from ATCC. The sequences of primers and fluorescent probe for coxsackievirus were adopted from previous publication (Oberste et al., 2010) and plaque titered virus stocks were used as standards for quantification. Primers and probes were manufactured by Integrated DNA Technologies (Coralville, IA). "
    [Show abstract] [Hide abstract] ABSTRACT: Viruses are major pathogens causing foodborne illnesses and are often present at low levels in foods, thus requiring sensitive techniques for their detection in contaminated foods. The lack of efficient culture methods for many foodborne viruses and the potential for multi-species viral contamination have driven investigation toward non-amplification based methods for virus detection and identification. A custom DNA microarray (FDA_EVIR) was assessed for its sensitivity in the detection and identification of low-input virus targets, human hepatitis A virus, norovirus, and coxsackievirus, individually and in combination. Modifications to sample processing were made to accommodate low input levels of unamplified virus targets, which included addition of carrier cDNA, RNase treatment, and optimization of DNase I-mediated target fragmentation. Amplification-free detection and identification of foodborne viruses were achieved in the range of 250 to 500 copies of virus RNA. Alternative data analysis methods were employed to distinguish the genotypes of the viruses particularly at lower levels of target input and the single probe-based analysis approach made it possible to identify a minority species in a multi-virus complex. The oligonucleotide array is shown to be a promising platform to detect foodborne viruses at low levels close to what are anticipated in food or environmental samples.
    Article · Mar 2016
Show more