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Duplex RT-PCR for simultaneous detection of hepatitis A and hepatitis E virus isolated from drinking water samples. 2000

N Jothikumar, and R Paulmurugan, and P Padmanabhan, and R B Sundari, and S Kamatchiammal, and K S Rao
National Environmental Engineering Research Institute, Chennai Zonal Laboratory, CSIR Complex, Taramani P.O., Chennai 600 113, India.

A duplex reverse transcription polymerase chain reaction (RT-PCR) protocol for simultaneous detection of hepatitis A virus (HAV) and hepatitis E virus (HEV) in water samples has been developed and demonstrated. Both HAV and HEV were concentrated from drinking water samples through a one-step concentration protocol. Different cDNA could be produced in the RT step carried out with a random primer in a single reaction tube. Two different sets of primers specific for HAV-cDNA and HEV-cDNA were used for PCR amplification. Amplified DNA products representing HAV and HEV were identified by gel electrophoresis at 247 and 327 bp (base pair) sequences, respectively. Specific sets of primers amplified a single type of virus and no cross-reactivity of the primers was noticed in duplex RT-PCR. The protocol was used for direct isolation and detection of HAV and HEV from 23 water samples in urban areas of Chennai city. Out of these, nine water samples were positive for HAV, and three for HEV. All three samples positive for HEV were also positive for HAV. The test provides a rapid and economical means of water quality surveillance to specifically detect HAV and HEV.

UI MeSH Term Description Entries
D002947 Cities A large or important municipality of a country, usually a major metropolitan center. Municipalities,Towns
D004279 DNA, Viral Deoxyribonucleic acid that makes up the genetic material of viruses. Viral DNA
D004784 Environmental Monitoring The monitoring of the level of toxins, chemical pollutants, microbial contaminants, or other harmful substances in the environment (soil, air, and water), workplace, or in the bodies of people and animals present in that environment. Monitoring, Environmental,Environmental Surveillance,Surveillance, Environmental
D006507 Hepatovirus A genus of PICORNAVIRIDAE causing infectious hepatitis naturally in humans and experimentally in other primates. It is transmitted through fecal contamination of food or water. HEPATITIS A VIRUS is the type species. Hepatitis Virus, Infectious,Infectious Hepatitis Virus,Hepatitis Viruses, Infectious,Hepatoviruses,Infectious Hepatitis Viruses
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D012680 Sensitivity and Specificity Binary classification measures to assess test results. Sensitivity or recall rate is the proportion of true positives. Specificity is the probability of correctly determining the absence of a condition. (From Last, Dictionary of Epidemiology, 2d ed) Specificity,Sensitivity,Specificity and Sensitivity
D014881 Water Supply Means or process of supplying water (as for a community) usually including reservoirs, tunnels, and pipelines and often the watershed from which the water is ultimately drawn. (Webster, 3d ed) Supplies, Water,Supply, Water,Water Supplies
D016752 Hepatitis E virus A positive-stranded RNA virus species, causing enterically-transmitted non-A, non-B hepatitis (HEPATITIS E). Hepatitis E virus (strain Burma),Orthohepevirus A,Paslahepevirus,Paslahepevirus balayani,Paslahepeviruses
D020133 Reverse Transcriptase Polymerase Chain Reaction A variation of the PCR technique in which cDNA is made from RNA via reverse transcription. The resultant cDNA is then amplified using standard PCR protocols. Polymerase Chain Reaction, Reverse Transcriptase,Reverse Transcriptase PCR,PCR, Reverse Transcriptase,Transcriptase PCR, Reverse

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