Development of a Microtiter Plate Hybridization-Based PCR-Enzyme-Linked Immunosorbent Assay for Identification of Clinically Relevant Human Group A Rotavirus G and P Genotypes

Santos, Norma; Honma, Shinjiro; Timenetsky, María do Carmo Sampaio Tavares; Linhares, Alexandre C.; Ushijima, Hiroshi; Armah, George; Gentsch, Jon R.; Hoshino, Yasutaka

doi:10.1128/jcm.01361-07

Cited by 31 publications

(17 citation statements)

References 84 publications

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“…To obtain information on older G2 VP7 gene sequences, we analyzed an archival collection of stool samples isolated from pediatric patients hospitalized with gastroenteritis at the Children's National Medical Center (CNMC) in Washington, DC, from 1974 to 1991 (12,(33)(34)(35)(36)(37). Total RNA was extracted from 27 samples positive for G2P [4] RV, as determined by microtiter plate hybridizationbased PCR enzyme-linked immunosorbent assay (ELISA) (38), using TRIzol (Life Technologies). The 15 samples containing the highest quality RNA were selected for sequencing.…”

Section: Methodsmentioning

confidence: 99%

Molecular Epidemiology of Contemporary G2P[4] Human Rotaviruses Cocirculating in a Single U.S. Community: Footprints of a Globally Transitioning Genotype

McDonald

et al. 2014

J Virol

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Group IMPORTANCELittle is known about the diversity of cocirculating G2 rotaviruses and how their evolution may undermine the effectiveness of rotavirus vaccines. To expand our understanding of the potential genetic range exhibited by rotaviruses circulating in postvaccine communities, we analyzed part of a collection of rotaviruses recovered from pediatric patients in the United States from 2010 to 2011. Examining the genetic makeup of these viruses revealed they represented three segregated groups that did not exchange genetic material. The distinction between these three groups may be explained by three separate introductions. By comparing a specific gene, namely, VP7, of the recent rotavirus isolates to those from a collection recovered from U.S. children between 1974 and 1991 and other globally circulating rotaviruses, we were able to reconstruct the timing of events that shaped their ancestry. This analysis indicates that G2 rotaviruses are continuously evolving, accumulating changes in their genetic material as they infect new patients.

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Section: Methodsmentioning

confidence: 99%

Molecular Epidemiology of Contemporary G2P[4] Human Rotaviruses Cocirculating in a Single U.S. Community: Footprints of a Globally Transitioning Genotype

McDonald

et al. 2014

J Virol

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“…These include type-specific PCR (20), restriction fragment length polymorphism (24), sequence analysis (3,5,10), capture and primer extension (32), and hybridization to oligonucleotide probes (7,43,44). …”

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confidence: 99%

Detection and Genotyping of Human Rotavirus VP4 and VP7 Genes by Reverse Transcriptase PCR and Reverse Hybridization

et al. 2009

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Rotavirus infections can be diagnosed in stool samples by serological and molecular methods. We developed a novel reverse transcriptase PCR (RT-PCR) method for the amplification of rotavirus RNA and a reverse hybridization assay on a strip to detect amplimers and identify the specific G and P genotypes present in human stool specimens. An additional aim was to permit specific identification of the rotavirus G1P[8] strain, used in the Rotarix vaccine. Novel broad-spectrum PCR primers were developed for both VP4 and VP7, permitting the amplification of a wide range of rotavirus genotypes. Primer sets comprise mixtures of defined primer sequences. For the identification of G and P genotypes, two reverse hybridization strip assays were developed. Both the VP4 and the VP7 strip contain universal probes for the detection of VP4 and VP7 sequences, irrespective of the G or P genotype. The VP4 strip contains type-specific probes for P[4], P[6], P[8], P[9], and P[10]. The VP7 strip contains type-specific probes for G1, G2, G3, G4, G5, G6, G8, and G9. In addition, probes to distinguish between wild-type G1 and G1 vaccine strain sequences were present. Testing by analysis of multiple reference strains confirmed that both RT-PCR methods allowed the detection of a broad spectrum of genotypes. RT-PCR for VP7 was more sensitive than RT-PCR for VP4, but all samples identified as positive for rotavirus antigen by an enzyme-linked immunosorbent assay (ELISA) were also positive for both VP4 and VP7. The high specificity of the reverse hybridization method was confirmed by sequence analysis as well as by type-specific PCR, and the vaccine strain could also be specifically identified. The reverse hybridization method permits accurate identification of mixed infections with different genotypes. Rotavirus genotypes for which no type-specific probes were present on the strip were adequately identified by the universal detection probes. The assay was formally validated by analyses of specificity, sensitivity, precision, accuracy, and robustness. In a panel of 149 ELISA-positive stool samples, comparison with conventional type-specific RT-PCR methods revealed the superiority of the novel method, mainly in cases of mixed rotavirus infections. This novel method permits highly accurate detection and identification of human rotavirus infections in stool samples. This validated assay could be useful for large-scale epidemiological and clinical trials.

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“…RT-PCR with an enzyme-linked immunosorbent assay (ELISA) has been proposed for the detection of HAV as an efficient alternative to RT-PCR with Southern blot hybrid ization. RT-PCR-ELISA has been used to detect several pathogenic viruses, including alphavirus, rotavirus, and viroids from clinical and environmental samples (26,28,32). To date, few researchers have evaluated the utility of RT-PCR-ELISA for detecting a pathogenic virus from food samples (20,27).…”

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confidence: 99%

Evaluation of a Sensitive Reverse Transcription PCR–Enzyme- Linked Immunosorbent Assay for Detection of Hepatitis A Virus in Oysters (Saccostrea glomerata) on the East Coast of the Gulf of Thailand

Intamaso

Ketkhunthod

2014

Journal of Food Protection

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Hepatitis A virus (HAV) contamination in food can lead to major health problems. We developed a combination reverse transcription (RT) PCR method plus enzyme-linked immunosorbent assay (ELISA) to detect HAV in fresh oysters harvested along the east coast of the Gulf of Thailand. Viral nucleic acid was extracted via the glycine-arginine-polyethylene glycol method followed by RT-PCR amplification with specifically designed primers against HAV and an ELISA to detect the digoxigenin-labeled RT-PCR products. The ELISA in concert with the RT-PCR protocol further increased the detection sensitivity by 100-fold for the HAV genome and 10-fold in artificially contaminated oysters. The overall sensitivity of the RT-PCR in combination with the ELISA was 31.88 pg and 16 PFU/g, respectively. The ELISA increases the specificity of the RT-PCR assay for detecting naturally occurring HAV in oysters. This combined RT-PCR-ELISA approach is a practical and sensitive method for HAV detection and can be utilized in routine screening for HAV in shellfish.

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Development of a Microtiter Plate Hybridization-Based PCR-Enzyme-Linked Immunosorbent Assay for Identification of Clinically Relevant Human Group A Rotavirus G and P Genotypes

Cited by 31 publications

References 84 publications

Molecular Epidemiology of Contemporary G2P[4] Human Rotaviruses Cocirculating in a Single U.S. Community: Footprints of a Globally Transitioning Genotype

Molecular Epidemiology of Contemporary G2P[4] Human Rotaviruses Cocirculating in a Single U.S. Community: Footprints of a Globally Transitioning Genotype

Detection and Genotyping of Human Rotavirus VP4 and VP7 Genes by Reverse Transcriptase PCR and Reverse Hybridization

Evaluation of a Sensitive Reverse Transcription PCR–Enzyme- Linked Immunosorbent Assay for Detection of Hepatitis A Virus in Oysters (Saccostrea glomerata) on the East Coast of the Gulf of Thailand

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