Polymerase chain reaction amplification and typing of rotavirus nucleic acid from stool specimens

Abstract

The rotavirus gene segment coding for the major outer capsid glycoprotein vp7 was amplified directly from stool specimens by the polymerase chain reaction (PCR). Double-stranded RNA extracted from stool samples was used as the template for reverse transcription, which was followed immediately and in the same reaction mix with amplification, using the Taq polymerase. Various conditions were examined to optimize the yield of the amplified gene. The concentrations of MgCl2, dimethyl sulfoxide, and template RNA were critical. The choice of primer pairs allowed amplification of the entire segment or specific portions. By using type-specific primers derived from distinct regions on the gene, we devised a PCR typing method in which each human serotype virus produced a characteristic segment size, readily identifiable in agarose gels. The PCR typing method was applied to 10 rotavirus reference strains, including all 6 known human serotypes (serotypes 1, 2, 3, 4, 8, and 9), and to 34 stool specimens previously serotyped by an enzyme immunoassay with monoclonal antibodies. An absolute correlation was found between the molecular and serologic methods. In addition, 14 stool specimens nonserotypable by an enzyme immunoassay with monoclonal antibodies could be typed by the PCR method. Besides the application for rotavirus detection and typing directly from stools, the PCR method provides a rapid and efficient means of obtaining large quantities of cDNA suitable for sequencing, cloning, and other genetic studies, precluding the need for cell culture and virus purification.

Equine rotaviruses—Current understanding and continuing challenges

“…Classification into P types was more complex and generally required either P type specific monoclonal antibodies or the generation of reassortant viruses with identical G serotype genes but distinct P type genes. Serotyping has been largely replaced by genotyping (Gentsch et al, 1992;Gouvea et al, 1990). The nucleotide sequence of the VP7 gene correlates closely with G serotype designations.…”

Molecular characterization of a novel bovine group A rotavirus

“…In order to perform P typing, Bov4Com5, Bov4Com3, P1, P5, P10, con2, con3, pNCDV, pUK, and pB223 primers were synthesized as described by Isegawa et al (1993) and Gouvea et al (1994a). To perform G typing, sBeg9, End9(UK), DT6, HT8, and ET10 primers were synthesized as described by Gouvea et al (1990Gouvea et al ( , 1993Gouvea et al ( , 1994b. Subsequently, the genome segments encoding the VP7s of the detected BoRV-As were amplified by PCR, using the primers VP7-MS (sense, 5 0 -CRGARYTAGATATGTCAGAA-3 0 , 491-510) and VP7-MA (antisense, 5 0 -AAYGTTATGTC-CATYGGATT-3 0 , 563-544).…”

Molecular characterization of rotavirus isolated from alpaca ( Vicugna pacos ) crias with diarrhea in the Andean Region of Cusco, Peru

“…The RT reaction was run at 25 C for 5 min, followed by 42 C for 45 min and the cDNA held at 4 C. The cDNA was used as the template to amplify VP7 or VP6 coding genes using the Green master mix kit (Promega Madison, WI). Briefly, reactions contained Green master mix, cDNA template, and Beg 9 and End 9 (Table 1) as forward and reverse primer sets for VP7 coding sequence (Gouvea et al, 1990), and nuclease-free water in a 25 ml/reaction. The PCR reaction was run at 94 C for 5 min, followed by 30 cycles of 94 C for 1 min, 50 C for 1 min, and 72 C for 1 min.…”