Comparison of direct and indirect enzyme immunoassays with direct ultracentrifugation before electron microscopy for detection of rotaviruses

Hammond, Gregory; Ahluwalia, Gurmukh S.; Barker, Frederick G.; Horsman, Greg B.; Hazelton, Paul R.

doi:10.1128/jcm.16.1.53-59.1982

Cited by 37 publications

(15 citation statements)

References 11 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…In some EIAs, however, the detector antibody is not conjugated to an enzyme. In these instances, a third, "anti-antibody" or indicator antibody which is enzyme conjugated is added (144,167,318,421). Often the third antibody has been used to obtain a more sensitive test or it has been used for typing.…”

Section: Laboratory Methods and Diagnosismentioning

confidence: 99%

Human viral gastroenteritis.

Christensen¹

1989

Clinical Microbiology Reviews

View full text Add to dashboard Cite

CHRISTENSEN propagated by using special cell types or techniques or both (320, 353, 372). In addition, a variety of in vitro detection systems, including enzyme immunoassays (149, 175, 333, 408), radioimmunoassays (137, 141, 181, 264), latex agglutination (140, 169), and nucleic acid hybridization techniques (116, 175), have been developed for their rapid detection. The detection and identification of these agents are important since viral gastroenteritis is the second most common clinical entity in developed countries, second only to viral upper respiratory tract illness (188). Worldwide, acute gastroenteritis and its associated dehydration afflicts almost 500 million children annually. In underdeveloped or developing countries, acute gastroenteritis, including viral gastroenteritis, is the leading cause of death of children under the age of 4 years (360). Since 1980, a wealth of literature has developed covering many aspects of these viruses and the infections they cause, including their molecular biology, epidemiology, immunology, and clinical features. In addition, detection tests for routine use have been developed for rotaviruses (310) and fecal adenoviruses (333) and experimental rotavirus vaccines have been developed (184, 377, 379, 380). Of these viruses, rotaviruses are the most common known cause of viral gastroenteritis in infants and young children, with the fastidious fecal adenoviruses most likely being the second most common cause in this age group (301, 369). Calici-, astro-, and coronaviruses are probably responsible for a minority of illness in the young age group (253, 302). In contrast, the Norwalk and Norwalk-like viruses have caused considerable numbers of outbreaks of gastroenteritis among older children, adolescents, and adults (136, 137, 189). ROTAVIRUSES Epidemiology Rotaviruses were first discovered in humans 15 years ago by Bishop et al. (18, 19) by the EM examination of duodenal

show abstract

Section: Laboratory Methods and Diagnosismentioning

confidence: 99%

Human viral gastroenteritis.

Christensen¹

1989

Clinical Microbiology Reviews

View full text Add to dashboard Cite

show abstract

“…Group A rotavirus is a double-stranded RNA virus which is recognized as a major cause of diarrheal disease in infants and young children living in all areas of the world. Since rotaviruses are difficult to cultivate directly from human fecal samples, rotavirus infections are generally identified by the direct detection of rotavirus antigens in diarrheal samples by immunoassay methods (2,12,13,15). However, there are situations in which assays with greater sensitivity are desirable in order to diagnose rotavirus infections and to detect low levels of viral shedding (19).…”

mentioning

confidence: 99%

Removal of inhibitory substances from human fecal specimens for detection of group A rotaviruses by reverse transcriptase and polymerase chain reactions

1990

View full text Add to dashboard Cite

A method was developed for the purification of rotavirus RNA from fecal extracts in order to permit the sensitive identification of group A rotavirus in fecal specimens by the polymerase chain reaction. Sequential reactions with reverse transcriptase and Taq polymerase with directed primers from rotavirus gene 6 yielded characteristic 259-base-pair fragments that were then visualized by silver stain on a polyacrylamide gel. As few as 500 genomic copies of purified rotavirus RNA could be detected in this manner. However, when the method was applied to fecal samples with added rotavirus virions, inhibition was noted in many of the fecal extracts which were tested. The inhibition could be reversed by dilution of the fecal extract, but sensitivity was also reduced by a corresponding dilutional factor. The inhibition was quantitatively removed by an added step in the extraction process that utilized chromatographic cellulose fiber powder (CF11 powder) to purify the rotavirus RNA during a series of rapid washing and elution steps. After CF11 purification, rotavirus RNA could be detected in experimental fecal samples at dilutions 1,000to 10,000-fold beyond the detection limits of standard techniques such as enzyme immunoassay and the direct visualization of RNA following polyacrylamide gel electrophoresis. Furthermore, following purification by CF11, rotavirus RNA could be detected in all of seven enzyme-linked immunosorbent assay-positive fecal samples obtained from a child with rotavirus gastroenteritis; when CF11 purification was not performed, rotavirus RNA could be detected in only four of these samples, even after the removal of inhibitors by dilution of the extracts. Large-scale identification of rotavirus in fecal specimens may be possible by use of CF11 purification of viral RNA prior to sequential reactions with reverse transcriptase and Taq polymerase in a modified polymerase chain reaction.

show abstract

“…La con-Parmi les tests immunoenzymatiques, Enzygnost et Rotazyme (Abbott) ont ~t~ les plus ~tudi~s (8,9,12) et se TABLEAU I R~cemment, plusieurs tests au latex ont ~t~ commer-cialis~s ~ la suite des travaux de Sanekata (10) et Haikala (2). Les avantages de telles techniques notamment I'acc~s & cette d~termination par des laboratoires non specialists, ont d~j& ~t~ d~crits (1,3,6,9). Les tests Rotalex et Slidex Rota-kit ont une sensibilit~ interm~diaire entre la ME et les tests immunoenzymatiques (6, 9) Slidex Rota-kit est I~g6rement plus sensible que Rotalex (84 % contre 72 %), fait d~j~ mentionn~ (5,9).…”

Section: Discussionunclassified