Detection of infectious hepatitis A virus by integrated cell culture/strand-specific reverse transcriptase-polymerase chain reaction

Jiang, Yunquan; Liao, Guoyang; Zhao, Wei; Sun, Maosheng; Qian, Yunliang; Bian, Chunjing; Jiang, Shimin

doi:10.1111/j.1365-2672.2004.02413.x

Cited by 40 publications

(29 citation statements)

References 35 publications

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“…The use of ICC-PCR has been described for the detection of enteroviruses (65), hepatitis A virus (42,64), enteric adenovirus (46), and astrovirus (36). The integrated use of cell culture with PCR has demonstrated a wide distribution of infectious viruses in water sources, since it allows for the detection of non-CPE-producing enteric viruses (47,64).…”

Section: Detection Of Viruses By Icc-pcrmentioning

confidence: 99%

“…During infection, the viral genome is transcribed to mRNA or another intermediary in the host cell which is eventually used for synthesis of viral proteins or replication of the The detection of these intermediaries in the host cell during infection is a clear indication that the virus is replicating in the host cell and that it is infectious. In the detection of a positive-strand RNA virus, the primer used is complementary to the sequence of the negative strand; the negative strand is transcribed to cDNA and then amplified by PCR (42). During cell infection with a virus such as HAV, the positive strand is transcribed to a negative strand in the host cell.…”

Section: Detection Of Viruses By Icc-pcrmentioning

confidence: 99%

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Application of PCR-Based Methods To Assess the Infectivity of Enteric Viruses in Environmental Samples

Rodríguez

Pepper

Gerba

2009

Appl Environ Microbiol

174

151

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Section: Detection Of Viruses By Icc-pcrmentioning

confidence: 99%

Section: Detection Of Viruses By Icc-pcrmentioning

confidence: 99%

Application of PCR-Based Methods To Assess the Infectivity of Enteric Viruses in Environmental Samples

Rodríguez

Pepper

Gerba

2009

Appl Environ Microbiol

174

151

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“…These strategies include using multiple PCR sites (41), longer amplicons (11,39,41), and immunocapture PCR (41). One method that has shown promise in selectively detecting infective RNA viruses is integrated cell culture/strand-specific reverse transcription-PCR (7,18) though, again, this method includes a culture-based step that is not available for many viruses.…”

mentioning

confidence: 99%

Quantitative PCR for Determining the Infectivity of Bacteriophage MS2 upon Inactivation by Heat, UV-B Radiation, and Singlet Oxygen: Advantages and Limitations of an Enzymatic Treatment To Reduce False-Positive Results

Pecson

Martin

Kohn

2009

Appl Environ Microbiol

158

168

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Health risks posed by waterborne viruses are difficult to assess because it is tedious or impossible to determine the infectivity of many viruses. Recent studies hypothesized that quantitative PCR (qPCR) could selectively quantify infective viruses if preceded by an enzymatic treatment (ET) to reduce confounding false-positive signals. The goal of this study was to determine if ET with qPCR (ET-qPCR) can be used to accurately quantify the infectivity of the human viral surrogate bacteriophage MS2 upon partial inactivation by three treatments (heating at 72°C, singlet oxygen, and UV radiation). Viruses were inactivated in buffered solutions and a lake water sample and assayed with culturing, qPCR, and ET-qPCR. To ensure that inactivating genome damage was fully captured, primer sets that covered the entire coding region were used. The susceptibility of different genome regions and the maximum genomic damage after each inactivating treatment were compared. We found that (i) qPCR alone caused false-positive results for all treatments, (ii) ET-qPCR significantly reduced (up to >5.2 log units) but did not eliminate the false-positive signals, and (iii) the elimination of false-positive signals differed between inactivating treatments. By assaying the whole coding region, we demonstrated that genome damage only partially accounts for virus inactivation. The possibility of achieving complete accordance between culture-and PCR-based assays is therefore called into doubt. Despite these differences, we postulate that ET-qPCR can track infectivity, given that decreases in infectivity were always accompanied by dose-dependent decreases in ET-qPCR signal. By decreasing false-positive signals, ET-qPCR improved the detection of infectivity loss relative to qPCR.Water-and food-borne viruses are a major worldwide source of gastroenteritis, and thus the detection and inactivation of infective viruses are important public health priorities. Cell culture can be employed to quantify the infectivity of certain viruses. However, culturing can take days to weeks to yield results, and many viruses important to public health, e.g., hepatitis A and noroviruses, are either difficult to culture or are nonculturable (3,19,38). Immunological and spectrometryand microscopy-based methods have also been developed, but none of them has provided a satisfactory alternative (5,26,43,45).The advent of PCR and quantitative PCR (qPCR) in environmental microbiology had raised hopes that these limitations would be overcome. While PCR lives up to its promise to provide rapid, sensitive, and specific detection of environmental viruses, its ability to differentiate infective from inactivated viruses has not been realized. This is mainly due to the persistence of genomes of inactivated viruses that remain either partially or fully intact. During PCR, intact genomic regions of inactivated viruses are amplified and produce confounding false-positive PCR signals (3,11,37,38,41). These false positives inhibit our ability to use qPCR to obtain quantitative inform...

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“…ICC-PCR detects virus infectivity faster than cell culture alone, but this method is still labor-intensive, and it is at least 2 or more days before results can be obtained. Researchers have used this technique for detection of infectious adenoviruses (20) and other enteric viruses (8,16,19,22,35,36).…”

mentioning

confidence: 99%

Use of Propidium Monoazide in Reverse Transcriptase PCR To Distinguish between Infectious and Noninfectious Enteric Viruses in Water Samples

Parshionikar

Laseke

Fout

2010

Appl Environ Microbiol

200

136

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Human enteric viruses can be present in untreated and inadequately treated drinking water. Molecular methods, such as the reverse transcriptase PCR (RT-PCR), can detect viral genomes in a few hours, but they cannot distinguish between infectious and noninfectious viruses. Since only infectious viruses are a public health concern, methods that not only are rapid but also provide information on the infectivity of viruses are of interest. The intercalating dye propidium monoazide (PMA) has been used for distinguishing between viable and nonviable bacteria with DNA genomes, but it has not been used to distinguish between infectious and noninfectious enteric viruses with RNA genomes. In this study, PMA in conjunction with RT-PCR (PMA-RT-PCR) was used to determine the infectivity of enteric RNA viruses in water. Coxsackievirus, poliovirus, echovirus, and Norwalk virus were rendered noninfectious or inactivated by treatment with heat (72°C, 37°C, and 19°C) or hypochlorite. Infectious or native and noninfectious or inactivated viruses were treated with PMA. This was followed by RNA extraction and RT-PCR or quantitative RT-PCR (qRT-PCR) analysis. The PMA-RT-PCR results indicated that PMA treatment did not interfere with detection of infectious or native viruses but prevented detection of noninfectious or inactivated viruses that were rendered noninfectious or inactivatedby treatment at 72°C and 37°C and by hypochlorite treatment. However, PMA-RT-PCR was unable to prevent detection of enteroviruses that were rendered noninfectious by treatment at 19°C. After PMA treatment poliovirus that was rendered noninfectious by treatment at 37°C was undetectable by qRT-PCR, but PMA treatment did not affect detection of Norwalk virus. PMA-RT-PCR was also shown to be effective for detecting infectious poliovirus in the presence of noninfectious virus and in an environmental matrix. We concluded that PMA can be used to differentiate between potentially infectious and noninfectious viruses under the conditions defined above.

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Detection of infectious hepatitis A virus by integrated cell culture/strand-specific reverse transcriptase-polymerase chain reaction

Cited by 40 publications

References 35 publications

Application of PCR-Based Methods To Assess the Infectivity of Enteric Viruses in Environmental Samples

Application of PCR-Based Methods To Assess the Infectivity of Enteric Viruses in Environmental Samples

Quantitative PCR for Determining the Infectivity of Bacteriophage MS2 upon Inactivation by Heat, UV-B Radiation, and Singlet Oxygen: Advantages and Limitations of an Enzymatic Treatment To Reduce False-Positive Results

Use of Propidium Monoazide in Reverse Transcriptase PCR To Distinguish between Infectious and Noninfectious Enteric Viruses in Water Samples

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