Rapid detection of human rotavirus using colorimetric nucleic acid sequence-based amplification (NASBA)–enzyme-linked immunosorbent assay in sewage treatment effluent

FEMS Microbiology Letters 210 (2002) 143^147 www.fems-microbiology.org Rapid detection of human rotavirus using colorimetric nucleic acid sequence-based ampli¢cation (NASBA)^enzyme-linked immunosorbent assay in sewage treatment e¥uent a a; Centre de Recherche STELA, De¤partement de Sciences des Aliments et de Nutrition, Universite¤ Laval, Laval, QC, Canada G1K 7P4 b De¤partement de Biochimie et Microbiologie, Universite¤ Laval, Laval, QC, Canada G1K 7P4 c Canadian Food Inspection Agency, Laboratory Services Division, Bldg 22, C.E.F., Ottawa, ON, Canada K1A 0C6 Received 7 January 2002; received in revised form 28 February 2002; accepted 5 March 2002 First published online 11 April 2002 Abstract A colorimetric nucleic acid sequence-based amplification^enzyme-linked immunosorbent assay (NASBA-ELISA) was developed for rapid detection and identification of human rotavirus. Oligonucleotide primers targeting gene 9 encoding a serotype-specific antigen VP7 were selected and used for the amplification of viral RNA by the isothermal NASBA process, resulting in the accumulation of biotinylated RNA amplicons. Amplicons were hybridized with a specific amino-linked oligonucleotide probe covalently immobilized on microtiter plates. The DNA^RNA hybrids were colorimetrically detected by the addition of streptavidin^peroxidase conjugate and tetramethylbenzidine substrate. Using the NASBA-ELISA system, as little as 0.2 PFU (4U101 PFU ml31 ) and 15 PFU (3U103 PFU ml31 ) of rotavirus were detected within 6 h in spiked MQ water and sewage treatment effluent respectively. No interference was encountered in the amplification and detection of rotavirus in the presence of non-target RNA or DNA. Moreover, the presence of nontarget bacteria and virus does not generate any non-specific signal, confirming the specificity of the developed NASBA-ELISA system and its effectiveness in specifically detecting rotavirus. The NASBA-ELISA system offers several advantages in terms of sensitivity, rapidity and simplicity. This technique should be readily adaptable for detection of other RNA viruses in both foods and clinical samples. C 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Rotavirus; Nucleic acid sequence-based ampli¢cation ; Microplate hybridization 1. Introduction In recent years, viral gastroenteritis has become extremely common in humans worldwide. Since 1982, 40% of the con¢rmed food-borne outbreaks reported throughout the world have been of viral etiology. Over 110 known enteric viruses are excreted in human feces. However, only a relatively small number have been epidemiologically linked to viral disease, namely Norwalk, rotavirus and hepatitis A virus. In the USA, rotaviruses cause about 3.5 million infections every year, resulting in 70 000 hospitalizations, 75^125 deaths of children and annual losses of * Corresponding author. Tel. : +1 (418) 656-2131 ext. 6825 ; Fax : +1 (418) 656-3353. E-mail address : ismail.£ (I. Fliss). nearly $250 millions [1]. Epidemiological evidence suggests that rotaviruses may be transmitted to humans by various foods, such as shell¢sh and crops. A major source of contamination is treated or raw waste water into which rotaviruses are excreted in large numbers from infected persons and where it can persist for a long period [2]. As for most enteric viruses, rotaviruses implicated in food-borne illness are often found in very low concentrations in contaminated water samples since they are not able to multiply in vitro. The ability to detect traces of rotavirus contamination in water and related samples is essential in developing tools for the investigation and possible prevention of viral disease outbreaks. Traditional methods for the detection of rotaviruses in water include tissue culture techniques in which viruses are concentrated from water samples and allowed to multiply in sensitive cell cultures in which characteristic cytopathic e¡ects can be visualized. Although sensitive, these techniques remain cumbersome, 0378-1097 / 02 / $22.00 C 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII : S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 6 1 4 - 6 FEMSLE 10446 14-5-02 Julie Jean a , Burton Blais c , Andre¤ Darveau b , Isma|«l Fliss 144 J. Jean et al. / FEMS Microbiology Letters 210 (2002) 143^147 2. Materials and methods 2.1. Viral and bacterial strains The human rotavirus strain Wa, kindly provided by Dr. P. Payment (Institut Armand-Frappier, Montreal, QC, Canada), was propagated in MA-104 cells using previously published method [1] and stored in 1-ml fractions at 380‡C until use. Hepatitis A virus HM-175 (HAV) and Escherichia coli (ATCC 11775) were used to evaluate the speci¢city of the NASBA reaction. HAV was propagated in FRhK-4 cells as previously described [10]. E. coli was grown in tryptic soy broth and bacterial counts were determined by plating on tryptic soy agar. 2.2. Nucleic acids Puri¢ed yeast tRNA (Roche Diagnostic, Laval, QC, Canada) and E. coli total RNA were used to evaluate the impact of non-homologous nucleic acids on the speci¢city of the NASBA reaction. Total RNA was puri¢ed from E. coli using Trizol reagent (Gibco-BRL, Burlington, ON, Canada). 2.3. Titration of rotavirus by immuno£uorescence microscopy The viral titer was determined in 96-well microtiter plates containing a 24-h MA-104 cell culture (150 000 cells ml31 ) in maintenance medium containing 5% fetal calf serum. The viral suspension was treated with 20 U trypsin ml31 for 30 min at room temperature and diluted in maintenance medium. 25 Wl of each dilution and 200 Wl of maintenance medium were added to microtiter plate wells. The microplates were incubated at 37‡C for 24 h in 5% CO2 . The medium was discarded and 100 Wl of 80% (v/v) acetone was added, followed by 30 min of incubation at 4‡C. In emptied wells, 50 Wl per well of anti-rotavirus antibody (1:300, Accurate Chemical, Westbury, NY, USA) was added and incubated for 30 min at 37‡C. The microplates were washed ¢ve times with 200 Wl of phosphate-bu¡ered saline (PBS) and 50 Wl of £uorescein isothiocyanate-labeled anti-sheep IgG (H+L) antibody (1:3000, Sigma, Oakville, ON, Canada) was added, followed by incubation for 30 min at 37‡C. The microplate was then washed four times with 200 Wl of PBS, 50 Wl of glycerol/PBS (3:1) was added to each well and the microplate was sealed until observation by epi£uorescence microscopy. Each viral titer was done in quadruplicate and calculated using the Reed^Muench method [11]. 2.4. Primers and oligonucleotide probes A primer pair was selected from published rotavirus gene 9 nucleic acid sequences encoding the serotype-speci¢c antigen VP7 (GenBank accession number K02033), synthesized and gel-puri¢ed before use (Table 1). The reverse primer (Rota-2) bears the bacteriophage T7 RNA polymerase promoter binding region and preferred transcriptional initiati (...truncated)