Detection of enteroviruses in groundwater with the polymerase chain reaction

Abstract

Standard methods for the detection of enteroviruses in environmental samples involve the use of cell culture, which is expensive and time-consuming. The polymerase chain reaction (PCR) is an attractive method for the detection of enteroviruses in water because primary cell culture is not needed and the increased sensitivity of PCR allows detection of the low numbers of target DNAs and RNAs usually found in environmental samples. However, environmental samples often contain substances that inhibit PCR amplification of target DNA and RNA. Procedures that remove substances that interfere with the amplification process need to be developed if PCR is to be successfully applied to environmental samples. An RNA-PCR assay for the detection of enteroviruses in water was developed and used to test a variety of groundwater concentrates and humic acid solutions seeded with poliovirus type 1. The groundwater samples and humic acid solutions were treated with Sephadex G-50, Sephadex G-100, Sephadex G-200, Chelex-100 resin, and a mixed bed resin to remove PCR-inhibitory material from the samples. Sephadex G-100 in combination with Chelex-100 was found to be very effective in removing inhibitory factors for the detection of enteroviruses in groundwater concentrates by PCR. Viruses were detected in two of the groundwater concentrates by the RNA-PCR assay after treatment with Sephadex G-100 plus Chelex-100. This was confirmed by tissue culture, suggesting that the treatment protocol and, subsequently, the RNA-PCR assay are applicable for the detection of enteroviruses in environmental samples.

Detection of toxigenicVibrio choleraefrom environmental water samples by an enrichment broth cultivation-pit-stop semi-nested PCR procedure

“…In addition, the presence of various interfering substances, such as humic acids, metal ions and organic matter (Rossen et al 1992), may lead to false-negative results. To overcome these problems, methods to separate DNA from extracts containing humic acid substances (Tsai and Olson 1992), and ®ltration through chelating ion exchange resins to eliminate metal ions (Abbaszadegan et al 1993), have been developed. Immunomagnetic beads attached to speci-®c antibodies to assist in capturing and concentrating organisms prior to DNA extraction have also been used (Islam et al 1993).…”

Methods to detect infectious human enteric viruses in environmental water samples

“…Therefore, it is important to include controls for detection of PCR inhibition for each sample. The inhibitory effect can then be relieved or reduced by several approaches such as pre-dilution of the extracted nucleic acid prior to PCR reaction (Brooks et al, 2005;Hamza et al, 2009a), the use of some PCR additives (Demeke and Adams, 1992;Kreader, 1996), removal of inhibitors during nucleic acid purification (Braid et al, 2003) and the use of some polymeric adsorbents (Abbaszadegan et al, 1993;Koonjul et al, 1999;Schriewer et al, 2011).…”

Detection of Enteroviruses and Mammalian Reoviruses in Korean Environmental Waters

“…Enteric viruses are transmitted in high numbers via the fecaloral route and are closely related with waterborne viral diseases. Enteric viruses have been found in rivers, groundwater, and even in treated drinking water (1,6) and are recognized as major public health hazards. They are known to cause various human illnesses such as meningitis, encephalitis, hepatitis, respiratory illnesses, gastroenteritis, and skin rashes, and include the polioviruses, coxsackieviruses, rotaviruses, hepatitis A virus (HAV), noroviruses, adenoviruses, and reoviruses (3,8,14).…”

“…The total culturable virus assay (TCVA) has been widely used as a standard method for the detection and quantification of infectious enteric viruses from environmental waters. A PCR-based assay is a powerful technique for detecting target RNA or DNA and could reduce the cost and time, and increase the detection sensitivity of a monitoring program (1,9). However, a variety of inorganic and organic inhibitors in water samples can affect the PCR results and amplify nucleic acids from noninfectious viruses.…”