Quantitative one-step RT-PCR assay for rapid and sensitive identification and titration of polioviruses in clinical specimens

Laassri, Majid; DiPiazza, Anthony; Bidzhieva, Bella; Zagorodnyaya, Tatiana; Chumakov, Konstantin

doi:10.1016/j.jviromet.2012.12.015

Cited by 12 publications

(16 citation statements)

References 12 publications

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“…Similar to our findings, the study also showed a significantly higher quantity of the vaccine virus detected by the multiplex PCR in culture positive samples compared to samples which were culture negative . Another study by Laassri et al, also found a quantitative multiplex real‐time PCR assay to be highly sensitive and specific compared to culture for Sabin poliovirus detection in clinical samples …”

Section: Discussionsupporting

confidence: 90%

“…In contrast, the turn‐around time was 10‐12 h for singleplex real‐time PCR and only 6‐7 h for the one‐step multiplex real‐time PCR. The substantial amount of time saved using real‐time PCR assays could be crucial in the earlier detection of outbreaks in the post‐eradication era, thus, giving sufficient time to prevent widespread transmission of polioviruses …”

Section: Discussionmentioning

confidence: 99%

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Comparison of culture, single and multiplex real‐time PCR for detection of Sabin poliovirus shedding in recently vaccinated Indian children

Giri

Rajan

Kumar

et al. 2017

Journal of Medical Virology

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Although, culture is considered the gold standard for poliovirus detection from stool samples, real‐time PCR has emerged as a faster and more sensitive alternative. Detection of poliovirus from the stool of recently vaccinated children by culture, single and multiplex real‐time PCR was compared. Of the 80 samples tested, 55 (68.75%) were positive by culture compared to 61 (76.25%) and 60 (75%) samples by the single and one step multiplex real‐time PCR assays respectively. Real‐time PCR (singleplex and multiplex) is more sensitive than culture for poliovirus detection in stool, although the difference was not statistically significant.

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Section: Discussionsupporting

confidence: 90%

Section: Discussionmentioning

confidence: 99%

Comparison of culture, single and multiplex real‐time PCR for detection of Sabin poliovirus shedding in recently vaccinated Indian children

Giri

Rajan

Kumar

et al. 2017

Journal of Medical Virology

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“…external control, which we found useful for quantitation (23). We developed our assay on the basis of S1, S2, and S3 primers and probes developed by the CDC because these have a track record of use and therefore this assay can be compared to previous studies.…”

Section: Discussionmentioning

confidence: 99%

Kinetics of Poliovirus Shedding following Oral Vaccination as Measured by Quantitative Reverse Transcription-PCR versus Culture

Taniuchi

Begum

et al. 2015

J Clin Microbiol

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Amid polio eradication efforts, detection of oral polio vaccine (OPV) virus in stool samples can provide information about rates of mucosal immunity and allow estimation of the poliovirus reservoir. We developed a multiplex one-step quantitative reverse transcription-PCR (qRT-PCR) assay for detection of OPV Sabin strains 1, 2, and 3 directly in stool samples with an external control to normalize samples for viral quantity and compared its performance with that of viral culture. We applied the assay to samples from infants in Dhaka, Bangladesh, after the administration of trivalent OPV (tOPV) at weeks 14 and 52 of life (on days 0 [pre-OPV], ؉4, ؉11, ؉18, and ؉25 relative to vaccination). When 1,350 stool samples were tested, the sensitivity and specificity of the quantitative PCR (qPCR) assay were 89 and 91% compared with culture. A quantitative relationship between culture ؉ / qPCR ؉ and culture ؊ /qPCR ؉ stool samples was observed. The kinetics of shedding revealed by qPCR and culture were similar. qPCR quantitative cutoffs based on the day ؉11 or ؉18 stool samples could be used to identify the culture-positive shedders, as well as the long-duration or high-frequency shedders. Interestingly, qPCR revealed that a small minority (7%) of infants contributed the vast majority (93 to 100%) of the total estimated viral excretion across all subtypes at each time point. This qPCR assay for OPV can simply and quantitatively detect all three Sabin strains directly in stool samples to approximate shedding both qualitatively and quantitatively. Both immunity to paralytic poliomyelitis and reduction of transmission of wild-type and vaccine-derived polioviruses are critical in polio eradication efforts (1, 2). Oral polio vaccine (OPV) is widely utilized in low-income settings for ease of delivery, low cost, and potential for mucosal immunity. OPV immunogenicity is imperfect (3), however, and shedding of vaccine strains further complicates the poliovirus reservoir. Measurement of poliovirus in stool samples can thus be useful in evaluating mucosal immunogenicity (4), in estimating the transmission intensities of vaccine-derived virus, and for surveillance after polio eradication (5).Poliovirus culture is still considered the gold standard procedure but requires up to two 7-day passages in two cell lines, followed by molecular or antigenic methods to differentiate and type the poliovirus isolates (6). This process is time-consuming and greatly diminishes the number of stool samples that can be surveyed. Furthermore, subcultured isolates are not always representative of the viruses present in the original stool sample and results are usually not quantitated by titer determination (7,8). PCR methods are therefore attractive in terms of cost, time, the ability to subtype, and quantitation (9-12). PCR for poliovirus has been found to be severalfold more sensitive than poliovirus isolation from cell culture (13) and has also been found to be more sensitive with cerebrospinal fluid, throat swabs, or stool samples (14-17). However, s...

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“…This low sensitivity might reflect intrinsic properties of degenerate primers and probes that use inosine and modified nucleotides to detect diverse nucleotide sequences conserved among the 3 types of PV in the VP1 coding region (7-10). With nondegenerate primers, highly sensitive rRT-PCR systems to detect PV vaccine strains have been developed (6,11). VDPV strains could also be detected with a recently developed rRT-PCR system (12).…”

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confidence: 99%

Development of an Efficient Entire-Capsid-Coding-Region Amplification Method for Direct Detection of Poliovirus from Stool Extracts

et al. 2015

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Laboratory diagnosis has played a critical role in the Global Polio Eradication Initiative since 1988, by isolating and identifying poliovirus (PV) from stool specimens by using cell culture as a highly sensitive system to detect PV. In the present study, we aimed to develop a molecular method to detect PV directly from stool extracts, with a high efficiency comparable to that of cell culture. We developed a method to efficiently amplify the entire capsid coding region of human enteroviruses (EVs) including PV. cDNAs of the entire capsid coding region (3.9 kb) were obtained from as few as 50 copies of PV genomes. PV was detected from the cDNAs with an improved PV-specific real-time reverse transcription-PCR system and nucleotide sequence analysis of the VP1 coding region. For assay validation, we analyzed 84 stool extracts that were positive for PV in cell culture and detected PV genomes from 100% of the extracts (84/84 samples) with this method in combination with a PV-specific extraction method. PV could be detected in 2/4 stool extract samples that were negative for PV in cell culture. In PV-positive samples, EV species C viruses were also detected with high frequency (27% [23/86 samples]). This method would be useful for direct detection of PV from stool extracts without using cell culture. Laboratory diagnosis has played a critical role in the Global Polio Eradication Initiative since 1988, by detecting and identifying poliovirus (PV). To date, PV has been detected by isolating the virus, using cell culture systems, from stool specimens from acute flaccid paralysis (AFP) cases, including polio and other paralysis cases, in the World Health Organization (WHO) Global Polio Laboratory Network (1, 2). The advantages of the cell culture-based procedure are (i) minimal equipment requirements, (ii) high sensitivity (detection limit of 1 infectious unit containing 50 to 1,000 virions) (3), and, importantly, (iii) biological amplification of PV to high titers (about 10 6 50% cell culture infectious dose [CCID 50 ] or 10 8 to 10 9 viral genome copies per l of cell lysate) for subsequent nucleotide sequence analysis of the capsid protein VP1 coding region, which is required for final identification and molecular epidemiological analysis of PV strains. Sequencing is essential for classifying PV isolates into vaccine strains, wild-type strains, and circulating vaccine-derived PV (VDPV) strains and for determining the necessity of additional vaccination plans by identifying virus reservoirs, along with molecular epidemiological methods (4). A major disadvantage of the cell culture system is the timeliness of reporting; it takes 10 days to confirm that a sample is negative for PV (2). To improve the efficiency of PV detection and identification, including the timeliness, the development of methods for direct detection of PV has been encouraged by WHO (http://www.polioeradication.org /Research/Grantsandcollaboration.aspx). Such direct detection methods must provide efficient PV detection, comparable to that of cell cultur...

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Quantitative one-step RT-PCR assay for rapid and sensitive identification and titration of polioviruses in clinical specimens

Cited by 12 publications

References 12 publications

Comparison of culture, single and multiplex real‐time PCR for detection of Sabin poliovirus shedding in recently vaccinated Indian children

Comparison of culture, single and multiplex real‐time PCR for detection of Sabin poliovirus shedding in recently vaccinated Indian children

Kinetics of Poliovirus Shedding following Oral Vaccination as Measured by Quantitative Reverse Transcription-PCR versus Culture

Development of an Efficient Entire-Capsid-Coding-Region Amplification Method for Direct Detection of Poliovirus from Stool Extracts

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