Real-time RT-PCR assays to differentiate wild-type group A rotavirus strains from Rotarix®and RotaTeq®vaccine strains in stool samples

Background.Group A rotavirus (RVA) infection is the major cause of acute gastroenteritis (AGE) in young children worldwide. Introduction of two live-attenuated rotavirus vaccines, RotaTeq® and Rotarix®, has dramatically reduced RVA associated AGE and mortality in developed as well as in many developing countries. High-throughput methods are needed to genotype rotavirus wild-type strains and to identify vaccine strains in stool samples. Quantitative RT-PCR assays (qRT-PCR) offer several advantages including increased sensitivity, higher throughput, and faster turnaround time.Methods.In this study, a one-step multiplex qRT-PCR assay was developed to detect and genotype wild-type strains and vaccine (Rotarix® and RotaTeq®) rotavirus strains along with an internal processing control (Xeno or MS2 RNA). Real-time RT-PCR assays were designed for VP7 (G1, G2, G3, G4, G9, G12) and VP4 (P[4], P[6] and P[8]) genotypes. The multiplex qRT-PCR assay also included previously published NSP3 qRT-PCR for rotavirus detection and Rotarix® NSP2 and RotaTeq® VP6 qRT-PCRs for detection of Rotarix® and RotaTeq® vaccine strains respectively. The multiplex qRT-PCR assay was validated using 853 sequence confirmed stool samples and 24 lab cultured strains of different rotavirus genotypes. By using thermostablerTthpolymerase enzyme, dsRNA denaturation, reverse transcription (RT) and amplification (PCR) steps were performed in single tube by uninterrupted thermocycling profile to reduce chances of sample cross contamination and for rapid generation of results. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments.Results.The VP7 qRT-PCRs exhibited 98.8–100% sensitivity, 99.7–100% specificity, 85–95% efficiency and a limit of detection of 4–60 copies per singleplex reaction. The VP7 qRT-PCRs exhibited 81–92% efficiency and limit of detection of 150–600 copies in multiplex reactions. The VP4 qRT-PCRs exhibited 98.8–100% sensitivity, 100% specificity, 86–89% efficiency and a limit of detection of 12–400 copies per singleplex reactions. The VP4 qRT-PCRs exhibited 82–90% efficiency and limit of detection of 120–4000 copies in multiplex reaction.Discussion.The one-step multiplex qRT-PCR assay will facilitate high-throughput rotavirus genotype characterization for monitoring circulating rotavirus wild-type strains causing rotavirus infections, determining the frequency of Rotarix® and RotaTeq® vaccine strains and vaccine-derived reassortants associated with AGE, and help to identify novel rotavirus strains derived by reassortment between vaccine and wild-type strains.

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Diagnostic Accuracy of Seven Commercial Assays for Rapid Detection of Group A Rotavirus Antigens

Journal of Clinical Microbiology ◽

10.1128/jcm.01984-15 ◽

2015 ◽

Vol 53 (11) ◽

pp. 3670-3673 ◽

Cited By ~ 15

Author(s):

Jérôme Kaplon ◽

Céline Fremy ◽

Sylvie Pillet ◽

Lucile Mendes Martins ◽

Katia Ambert-Balay ◽

...

Keyword(s):

Diagnostic Accuracy ◽

Real Time ◽

Reverse Transcription ◽

Acute Gastroenteritis ◽

Rapid Detection ◽

Group A Rotavirus ◽

Reverse Transcription Pcr ◽

Group A ◽

Stool Samples ◽

Human Stool

Seven commercial immunochromatographic assays intended for the detection of group A rotavirus antigens in human stool samples were evaluated. These assays showed similar levels of diagnostic accuracy and were suitable for the detection of rotavirus in patients with acute gastroenteritis but missed some asymptomatic rotavirus shedding identified by real-time reverse transcription-PCR.

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Detection of SARS Coronavirus in Patients with Severe Acute Respiratory Syndrome by Conventional and Real-Time Quantitative Reverse Transcription-PCR Assays

Clinical Chemistry ◽

10.1373/clinchem.2003.023663 ◽

2004 ◽

Vol 50 (1) ◽

pp. 67-72 ◽

Cited By ~ 76

Author(s):

Leo L M Poon ◽

Kwok Hung Chan ◽

On Kei Wong ◽

Timothy K W Cheung ◽

Iris Ng ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Real Time ◽

N Gene ◽

Clinical Samples ◽

Rt Pcr ◽

Pcr Assay ◽

Reverse Transcription Pcr ◽

Copy Numbers ◽

Stool Samples ◽

Pcr Assays

Abstract Background: A novel coronavirus (CoV) was recently identified as the agent for severe acute respiratory syndrome (SARS). We compared the abilities of conventional and real-time reverse transcription-PCR (RT-PCR) assays to detect SARS CoV in clinical specimens. Methods: RNA samples isolated from nasopharyngeal aspirate (NPA; n = 170) and stool (n = 44) were reverse-transcribed and tested by our in-house conventional RT-PCR assay. We selected 98 NPA and 37 stool samples collected at different times after the onset of disease and tested them in a real-time quantitative RT-PCR specific for the open reading frame (ORF) 1b region of SARS CoV. Detection rates for the conventional and real-time quantitative RT-PCR assays were compared. To investigate the nature of viral RNA molecules in these clinical samples, we determined copy numbers of ORF 1b and nucleocapsid (N) gene sequences of SARS CoV. Results: The quantitative real-time RT-PCR assay was more sensitive than the conventional RT-PCR assay for detecting SARS CoV in samples collected early in the course of the disease. Real-time assays targeted at the ORF 1b region and the N gene revealed that copy numbers of ORF 1b and N gene sequences in clinical samples were similar. Conclusions: NPA and stool samples can be used for early diagnosis of SARS. The real-time quantitative RT-PCR assay for SARS CoV is potentially useful for early detection of SARS CoV. Our results suggest that genomic RNA is the predominant viral RNA species in clinical samples.

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Monitoring Shedding of Five Genotypes of RotaTeq Vaccine Viruses by Genotype-Specific Real-Time Reverse Transcription-PCR Assays

Journal of Clinical Microbiology ◽

10.1128/jcm.00035-18 ◽

2018 ◽

Vol 56 (6) ◽

Cited By ~ 4

Author(s):

Yuki Higashimoto ◽

Masaru Ihira ◽

Yu Miyazaki ◽

Ayumi Kuboshiki ◽

Sayaka Yoshinaga ◽

...

Keyword(s):

Real Time ◽

Reverse Transcription ◽

Cross Reactivity ◽

Rt Pcr ◽

Fecal Shedding ◽

Reverse Transcription Pcr ◽

Coefficients Of Variation ◽

Long Time ◽

Stool Samples ◽

Pcr Assays

ABSTRACTRotaTeq (RV5) is a widely used live attenuated pentavalent rotavirus (RV) vaccine. Although fecal shedding of RV vaccine strains persists for long time periods, it is unclear how each vaccine strain replicates in intestinal tissue and is excreted in stool. To examine this issue, we established RV5 genotype-specific real-time reverse transcription-PCR (RT-PCR) assays. Five real-time RT-PCR assays were designed for the VP7 gene in genotypes G1, G2, G3, G4, and G6. All assays exhibited excellent linearity, and the detection limit was 1 infectious unit (IU)/reaction for G2, G4, and G6 and 10 IUs/reaction for G1 and G3. No cross-reactivity was observed among G genotypes. The inter- and intra-assay coefficients of variation were less than 3%. The assays were used to examine 129 stool samples collected from eight infants who received RV5. In cases 1 and 2, who received three rounds of vaccination, RV shedding decreased gradually with the number of vaccinations. G1 and G6 shedding appeared to be predominant in comparison to shedding of the other genotypes. Patterns of fecal shedding of the five genotypes of vaccine viruses differed between the eight vaccine recipients. RV5 genotype-specific real-time RT-PCR assays will be useful to study the molecular biology of RV5 replication in infants and experimental animals.

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Novel Human Astroviruses: Prevalence and Association with Common Enteric Viruses in Undiagnosed Gastroenteritis Cases in Spain

Viruses ◽

10.3390/v11070585 ◽

2019 ◽

Vol 11 (7) ◽

pp. 585 ◽

Cited By ~ 5

Author(s):

Diem-Lan Vu ◽

Aurora Sabrià ◽

Nuria Aregall ◽

Kristina Michl ◽

Virginia Rodriguez Garrido ◽

...

Keyword(s):

Real Time ◽

Acute Gastroenteritis ◽

Enteric Viruses ◽

Epidemiological Studies ◽

Diagnostic Methods ◽

Rt Pcr ◽

Routine Diagnostics ◽

Human Astroviruses ◽

Stool Samples ◽

Pcr Assays

A remarkable percentage of acute gastroenteritis cases remain etiologically undiagnosed. The aim of the study was to determine the prevalence of common and emerging enteric viruses, such as novel human astroviruses, among undiagnosed samples from children with acute gastroenteritis. Epidemiological studies for novel human astroviruses are still scarce. Stool samples collected over two consecutive winter seasons (2016–2017) from children with gastroenteritis in Spain, which were negative for bacteria, rotavirus, and adenovirus by routine diagnostics were screened by real-time RT-PCR assays for the presence of classical and novel astrovirus, rotavirus, norovirus GI and GII, sapovirus, and adenovirus. Overall, 220/384 stool samples (57.3%) were positive for at least one virus. Co-infections were identified in 21% of cases. Among a total of 315 viruses identified, adenovirus was the most prevalent (n = 103), followed by rotavirus (n = 51), sapovirus (n = 50), classical astrovirus (n = 43), novel astroviruses (n = 42), and norovirus (n = 26). Novel astroviruses were present in 13.3% of virus-positive cases. Most novel astroviruses were found in children <2-year-old (30/39 children, 77%, p = 0.01) and were found in co-infection (66%). Only classical astroviruses demonstrated significant differences in the Cq values during mono-infections compared to co-infections. In conclusion, common enteric viruses may be frequently found in children with undiagnosed gastroenteritis, indicating the need to implement more sensitive diagnostic methods. Novel astroviruses circulate in the community and could be the cause of gastroenteritis among young children.

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Molecular Detection of Group A Rotavirus in under Five Years Old Children with Acute Diarrhoea Admitted to Yangon Children’s Hospital

Myanmar Health Sciences Research Journal ◽

10.34299/mhsrj.00927 ◽

2019 ◽

Vol 31 (1) ◽

pp. 87-92

Keyword(s):

Acute Diarrhoea ◽

Enzyme Linked Immunosorbent Assay ◽

Viral Gastroenteritis ◽

Cross Sectional ◽

Under Five ◽

Group A Rotavirus ◽

Rotavirus Vaccination ◽

Group A ◽

Stool Samples ◽

Considerable Morbidity

Rotaviruses are regarded as the most common cause of viral gastroenteritis and are responsible for considerable morbidity and mortality among children especially under five years of age worldwide. In developing countries like Myanmar, where diarrhoea is in the priority childhood disease, rotavirus surveillance and detection of rotavirus genotypes are utmost important. A hospital-based, cross-sectional descriptive study was conducted at Yangon Children‟s Hospital among under five children admitted for acute diarrhoea from January to October 2016. This study includes detection of Group A rotavirus antigen by commercial enzyme-linked immunosorbent assay (ELISA) and genotyping by multiplex RT-PCR. From a total of 488 collected samples, rotavirus antigen was detected in 219 samples (45%). Rotavirus diarrhoea was most common among the age of 6-11 months (38.8%) followed by 12-23 months (37.9%). The results showed that boys were more commonly affected than girls. Detection of rotavirus positivity was peak in February (57.6 %). Out of 219 stool samples with positive ELISA result, 40 stool samples with high optical density value were proceeded for further determination of G and P genotypes. Regarding distribution of G genotypes, the most common G genotype was G9 which comprised 45%, and that of P genotype was P[8] which comprised 92.5%. Regarding combination of G and P genotypes, the most frequent combination is G9P[8], and it constituted 42.5%. Untypable genotypes were seen in 30% of G and 2.5% of P typing. As rotavirus infection can be prevented by vaccine, WHO recommended that rotavirus vaccination should be included in national immunization program especially in countries where prevalence of rotavirus is high. The distribution of G and P genotypes is important in consideration of appropriate vaccine in pre-vaccination and evaluation of effectiveness of vaccine in post-vaccination period. Therefore, the information on currently circulating genotypes of rotavirus in this study will serve as valuable data for vaccination programme.

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Differing Effects of Standard and Harsh Nucleic Acid Extraction Procedures on Diagnostic Helminth Real-Time PCRs Applied to Human Stool Samples

Pathogens ◽

10.3390/pathogens10020188 ◽

2021 ◽

Vol 10 (2) ◽

pp. 188

Author(s):

Tanja Hoffmann ◽

Andreas Hahn ◽

Jaco J. Verweij ◽

Gérard Leboulle ◽

Olfert Landt ◽

...

Keyword(s):

Nucleic Acid ◽

Real Time ◽

Real Time Pcr ◽

Acid Extraction ◽

Nucleic Acid Extraction ◽

Pcr Assay ◽

Bead Beating ◽

Stool Samples ◽

Pcr Assays ◽

Human Stool

This study aimed to assess standard and harsher nucleic acid extraction schemes for diagnostic helminth real-time PCR approaches from stool samples. A standard procedure for nucleic acid extraction from stool and a procedure including bead-beating as well as proteinase K digestion were compared with group-, genus-, and species-specific real-time PCR assays targeting helminths and nonhelminth pathogens in human stool samples. From 25 different in-house and commercial helminth real-time PCR assays applied to 77 stool samples comprising 67 historic samples and 10 external quality assessment scheme samples positively tested for helminths, higher numbers of positive test results were observed after bead-beating-based nucleic acid extraction for 5/25 (20%) real-time PCR assays irrespective of specificity issues. Lower cycle threshold values were observed for one real-time PCR assay after the standard extraction scheme, and for four assays after the bead-beating-based scheme. Agreement between real-time PCR results after both nucleic acid extraction strategies according to Cohen’s kappa ranged from poor to almost perfect for the different assays. Varying agreement was observed in eight nonhelminth real-time PCR assays applied to 67 historic stool samples. The study indicates highly variable effects of harsh nucleic acid extraction approaches depending on the real-time PCR assay used.

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