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PubMed1999

Serotypes of human rotaviruses in 7 regions of Japan from 1984 to 1997.

Human rotavirus (HRV) serotypes were studied from diarrheal stool specimens in children in 7 regions of Japan (Sapporo, Tokyo, Maizuru, Osaka, Kagawa, Kurume, and Saga) from 1984 to 1997 by enzyme immunoassay (EIA) with serotype-specific monoclonal antibodies against serotypes 1, 2, 3, and 4. In addition, reverse transcription-polymerase chain reaction (RT-PCR) was conducted for analysis of "others" which included nonserotypable and mixed-serotype rotavirus specimens by EIA. In 3756 rotavirus-positive specimens, serotype 1 was detected in 2649 (70.5%), serotype 2 in 362 (9.6%), serotype 3 in 232 (6.2%) and serotype 4 in 196 (5.2%). Overall, serotype 1 was predominant from 1984 to 1997, although there were a few cases in which serotype 2, 3 and 4 became predominant based on area and year. The frequency of serotype 1 has gradually increased since 1993. Twenty two, 2, 3 and 1 among 57 specimens of "others" by EIA from Tokyo, Maizuru, Sapporo and Kurume in 1995-1996 and 1996-1997 were determined as serotypes 1, 2, 3, and 9 by RT-PCR, respectively.

Child↗
PubMed1969

Determination and analysis of Actinomyces israelii serotypes by fluorescent-antibody procedures.

This study was undertaken to determine the antigenic relationships between serotypes of Actinomyces israelii with fluorescent-antibody (FA) procedures. In addition, the antigenic relationships between A. israelii and other members of the genus Actinomyces were studied by the same methods. Seven FA conjugates were used to determine the serological characteristics of 28 isolates believed to represent A. israelii, serotypes 1 and 2. The results showed that the lower dilutions of serotype 1 conjugates stained serotype 2 antigens; however, serotype 2 conjugates did not stain serotype 1 antigens. Serotype 1 conjugate could be made specific by adsorption. A. israelii serotype 1 conjugate cross-reacted also with A. naeslundii, but this cross-reaction could be eliminated by adsorption or dilution. Serotype 2 conjugate appeared to be specific for A. israelii serotype 2. Adsorption studies revealed antigenic variants among the various A. israelii serotype 1 and 2 isolates. However, all isolates could be identified by direct FA staining with appropriate conjugates. One isolate previously identified as A. israelii was shown, on the basis of FA studies, to be an A. naeslundii. A polyvalent diagnostic reagent was prepared which was specific for A. israelii serotypes 1 and 2. This reagent should find application in diagnostic and reference laboratories.

Actinomyces↗
PubMed1988

Subgroups, serotypes, and electrophoretypes of rotavirus isolated from children in Bangui, Central African Republic.

The subgroups and serotypes of 178 strains of rotavirus isolated from diarrheic and healthy children in Bangui, Central African Republic, during a 27-month period were determined by enzyme-linked immunosorbent assay. The subgroup was determined for 152 of the viral strains, 18.4% being subgroup I and 81.6% being subgroup II. Of the 143 strains which could be serotyped, 71.3% were serotype 1, 15.4% were serotype 2, and 13.3% were serotype 3. Serotypes 1 and 3 were detected throughout the study, while serotype 2 was detected only during 8 months. No serotype exhibited any special epidemiological properties. The serotypes were found to consist of three different electrophoretypes, two long ones (A and B) and a short one (C). All subgroup I, serotype 2 strains presented short electrophoretypes. Strains with identical long electrophoretypes A were either serotype 1 or serotype 3.

Central African Republic↗
PubMed1989

Temporal and geographical distributions of human rotavirus serotypes, 1983 to 1988.

Between 1983 and 1988, subgroups and serotypes were determined for 907 of 1,084 clinical specimens of rotaviruses collected in various countries of Europe, North and South America, Africa, and Asia. Enhanced enzyme immunoassays based on monoclonal antibodies specific for rotavirus proteins VP6 and VP7 were used. Significant differences in the prevalent serotypes were detected from year to year in the United Kingdom and Brazil and also in different countries during the same year. Throughout the study, rotavirus serotype 1 was detected most often (53.8%), followed in frequency by serotype 2 (17.8%), serotype 3 (12.1%), serotype 4 (11.1%), and serotypes other than 1 to 4 (5.1%). No individual serotype was found to predominate consistently in any one location. In the United Kingdom, rotavirus serotypes varied in prevalence in a regular but not predictable way. We suggest that a similar epidemiology might be found in other settings. Seventeen unusual strains were detected. Of these, five strains did not react with reference monoclonal antibodies specific for subgroup I and subgroup II, but they reacted with rotavirus group A-specific polyclonal and monoclonal antibodies; four strains were of subgroup II, serotype 2, and at least one had a "long" electropherotype; two strains were of subgroup I, serotype 2 with a long electropherotype; and one strain was of subgroup I, serotype 3. Five group C rotaviruses were detected.

Brazil↗
PubMed1993

An OspA serotyping system for Borrelia burgdorferi based on reactivity with monoclonal antibodies and OspA sequence analysis.

A total of 136 Borrelia burgdorferi sensu latu strains from various biological sources (ticks, human skin, and cerebrospinal fluid) and geographical sources (Europe and North America) were investigated by Western blot (immunoblot) with eight monoclonal antibodies against different epitopes of the outer surface protein A (OspA). On the basis of the differential reactivities of these monoclonal antibodies, seven OspA serotypes were defined. As determined by 16S rRNA sequence analysis, these serotypes correlated well with recently delineated genospecies: serotype 1 corresponds to B. burgdorferi sensu strictu, serotype 2 corresponds to group VS461, and serotypes 3 to 7 correspond to Borrelia garinii sp. nov. (G. Baranton, D. Postic, I. Saint Girons, P. Boerlin, J.-C. Piffaretti, M. Assous, and P. A. D. Grimont, Int. J. Syst. Bacteriol. 42:378-383, 1992). Antigenic differences were confirmed by partial sequence analysis of OspA of representatives of each serotype. Comparative sequence analysis suggested that serotype 5 OspA resulted from genetic recombination of serotype 4 and 6 ospA genes. Serotype 2 (group VS461) was most prevalent among European skin isolates (49 of 62 isolates). Among all B. garinii strains included in this study, serotype 6 was most frequently found in ticks and only rarely in human skin and cerebrospinal fluid, whereas serotypes 4 and 5 were isolated from patients but never from ticks. Our data suggest different pathogenic potentials and organotropisms of distinct OspA serotypes and raise the question of true antigenic variation among B. garinii strains.

Amino Acid Sequence↗
PubMed1993

Serotyping of Cryptococcus neoformans by using a monoclonal antibody specific for capsular polysaccharide.

The importance of epidemiological studies of cryptococcosis has increased since the beginning of the AIDS epidemic. Cryptococcus neoformans exists in two varieties defining four serotypes, Cryptococcus neoformans var. neoformans (serotypes A and D) and Cryptococcus neoformans var. gattii (serotypes B and C). The varieties are easy to distinguish by their differences of growth on diagnostic media. We propose here an easy serotyping method combining diagnostic media and a direct immunofluorescence assay with one monoclonal antibody (E1) specific for cryptococcal polysaccharide. The method was validated by the blinded testing of four to five reference strains of each serotype. Immunofluorescence patterns were characteristic of a given serotype provided that the variety of the strain had been defined before. For C. neoformans var. neoformans, a bright, homogeneous staining with several cell aggregates was characteristics of serotype A, whereas only a few serotype D cells were positive. For C. neoformans var. gattii, a completely negative isolate was serotype C, whereas the population of serotype B included a majority of negative cells but also included positive cells with a speckled pattern. The method was then used to serotype 156 clinical isolates from France and isolates from areas where C. neoformans var. gattii was endemic before the AIDS epidemic (13 strains from Rwanda and Zaire and 5 strains from Australia). The specificity of E1 was defined by its reactivity with various Cryptococcus spp. and analyzed according to the described cryptococcal antigenic factors. We conclude from this study that E1 provides a rapid and reliable means to serotype multiple isolates of C. neoformans.

AIDS-Related Opportunistic Infections↗
PubMed1993

Serologic analysis of human rotavirus serotypes P1A and P2 by using monoclonal antibodies.

Three human rotavirus (HRV) VP4 serotypes and one subtype have been described on the basis of a fourfold or an eightfold-or-greater difference in neutralization titer when tested with hyperimmune antisera to recombinant VP4 or VP8* (serotypes P1A, P1B, P2, and P3). To start to analyze the antigenic basis underlying serotype specificity, we produced a library of 13 VP4-specific neutralizing monoclonal antibodies (NMAbs) to two HRVs, the serotype P1A strain Wa and the serotype P2 strain ST3, and characterized the reactivity of these NMAbs with a panel of serotypically diverse HRV strains by neutralization assay and enzyme-linked immunosorbent assay (ELISA). We characterized the serotypic specificity of the NMAbs by using a fourfold or an eightfold-or-greater difference in titer against the homologous (i.e., immunogen) and heterologous strains as a criterion for serotype. Some ST3-derived NMAbs reacted specifically with serotype P2 HRVs by ELISA and/or neutralization assay, while some Wa-derived NMAbs reacted specifically by ELISA and/or neutralization assay with some or all serotype P1A HRVs. Other Wa- and ST3-derived NMAbs reacted with some or all serotype P1A and P2 HRV strains by neutralization assay and ELISA. Most NMAbs did not react with serotype P1B or P3 strains. In previous studies, three distinct operationally defined epitopes have been identified on VP4 by examining the reactivity patterns of selected antigenic variants of HRV strain KU. At least one of the NMAbs described here recognizes an epitope unrelated to these previously identified epitopes, since it neutralized both KU and its variants.

Animals↗
PubMed2000

Molecular analysis of CAP59 gene sequences from five serotypes of Cryptococcus neoformans.

The nucleotide sequences of CAP59 genes from five serotypes of Cryptococcus neoformans were analyzed for their phylogenetic relationships. Approximately 600-bp genomic DNA fragments of the CAP59 gene were amplified from each isolate by PCR and sequenced. The CAP59 nucleotide sequences of C. neoformans showed more than 90% similarity among the five serotypes. By phylogenetic analysis, their sequences were divided into three clusters: serotypes A and AD, serotypes B and C, and serotype D. In addition, the results of reduced amino acid sequences were similar to the nucleotide sequence data. These data revealed that serotype AD was genetically close to serotype A rather than serotype D, although it had been considered to be a mixed type of serotype A and D by serological analysis. Furthermore, the nucleotide sequences of the serotype B and C isolates of C. neoformans were very similar to each other. These results indicated that serotype B and C isolates belonging to C. neoformans var. gattii were genetically homogeneous and closely related. The molecular analysis of the CAP59 gene will provide useful information for the differentiation of serotypes of C. neoformans and for an understanding of their phylogenetic relationships.

Animals↗
PubMed1983

Comparative inhibition of cellular transcription by vesicular stomatitis virus serotypes New Jersey and Indiana: role of each viral leader RNA.

We compared the ability of the leader RNAs of the New Jersey and Indiana serotypes of vesicular stomatitis virus to inhibit transcription in infected host cells. The level of cellular RNA synthesis in cells infected with either serotype was drastically reduced by 5 h after infection. Studies with UV-inactivated virus demonstrated that shutoff of cellular RNA synthesis directly correlated with the ability of the infecting virus to transcribe its plus-stranded leader RNA. Although both serotypes inhibited cellular RNA synthesis, the Indiana serotype reduced synthesis to lower levels. In addition, an examination of the kinetics of leader RNA synthesis in vivo indicated that up to four times more leader RNA was produced in cells infected with the Indiana serotype than in those infected with the New Jersey serotype. However, in vivo studies also suggested that the leader RNA of the New Jersey serotype was a more efficient RNA inhibitor than was the Indiana serotype leader RNA. Although up to 2,900 copies of the leader RNA per cell could be detected in infected cells, only 550 copies of the Indiana and 100 copies of the New Jersey leader RNAs per cell were present in infected cells that were demonstrating 50% of the maximal inhibition of RNA synthesis. In an in vitro system, leader RNAs of both serotypes inhibited DNA-dependent transcription of the adenovirus late promoter and adenovirus-associated RNA genes, but the New Jersey serotype leader was also a better inhibitor in this reconstituted system. Data from the dose response of inhibition by each leader suggest that polymerase III transcription was more sensitive to inhibition by viral leaders than was polymerase II transcription. Polyadenylated viral mRNAs and the NS and N gene starts transcribed by both serotypes did not significantly inhibit transcription at levels at which the corresponding leader RNAs were inhibitory. Overall, our results strongly suggest a role for the plus-stranded leader RNAs of the New Jersey and Indiana serotypes of vesicular stomatitis virus in inhibiting cellular transcription in vivo. We discuss differences in the nucleotide sequences of the two leader RNAs in relation to their differences in biological activity and to potential regulatory sequences.

Animals↗
PubMed2005

Comparative sequence analysis of the hexon gene in the entire spectrum of human adenovirus serotypes: phylogenetic, taxonomic, and clinical implications.

The adenovirus (AdV) hexon constitutes the major virus capsid protein. The epitopes located on the hexon protein are targets of neutralizing antibodies in vivo, serve in the recognition by cytotoxic T cells, and provide the basis for the classification of adenoviruses into the 51 serotypes known to date. We have sequenced the entire hexon gene from human serotypes with incomplete or no sequence information available (n = 34) and performed a comparative analysis of all sequences. The overall sequence divergence between the 51 human serotypes ranged from 0.7 to 25.4% at the protein level. The sequence information has been exploited to assess the phylogeny of the adenovirus family, and protein distances between the six AdV species (A to F) and among individual serotypes within each species were calculated. The analysis revealed that the differences among serotypes within individual species range from 0.3 to 5.4% in the conserved regions (765 amino acids [aa]) and from 1.5 to 59.6% in the variable regions (154 to 221 aa). Serotypes of different species showed an expectedly greater divergence both in the conserved (5.9 to 12.3%) and variable (49.0 to 74.7%) regions. Construction of a phylogenetic tree revealed three major clades comprising the species B+D+E, A+F, and C, respectively. For serotypes 50 and 51, the original assignment to species B and D, respectively, is not in accordance with the hexon DNA and protein sequence data, which placed serotype 50 within species D and serotype 51 within species B. Moreover, the hexon gene of serotype 16, a member of species B, was identified as the product of recombination between sequences of species B and E. In addition to providing a basis for improved molecular diagnostics and classification, the elucidation of the complete hexon gene sequence in all AdV serotypes yields information on putative epitopes for virus recognition, which may have important implications for future treatment strategies permitting efficient targeting of any AdV serotype.

Adenovirus Infections, Human↗
PubMed1983

Serotypes of Neisseria meningitidis associated with an increased incidence of meningitis cases in the Hamilton area, Ontario, during 1978 and 1979.

The distribution of serotypes among strains of Neisseria meningitidis responsible for a marked increase of meningitis cases in the Hamilton area, Ontario, in 1978 and 1979 was determined. Twenty-six serogroup B and two serogroup W135 strains isolated from cerebrospinal fluid, blood, and skin of 28 patients were serotyped by agar gel double diffusion. Twenty-one (81%) of the group B strains were serotype 2b as judged by the formation of characteristic serotype precipitin bands with the specific anti-2996 (type 2b) serum. Fourteen of the serotype 2b strains also reacted with anti-77252 serum, which suggested that one strain or several closely related strains were mainly responsible for the increase in meningitis during the 2-year period. Examination of the outer membrane complexes (OMC) of the strains by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) revealed that all 21 of the serotype 2b strains contained the class 2 protein (molecular weight 41500) which is known to be the site of the serotype 2b determinant. Further characterization of the serotype 2b, 77252 strains by enzyme-linked immunosorbent assays (ELISA) and SDS-PAGE suggested that the 77252 determinant was present in the class 1 proteins of these strains. The serotype 2b containing strains were isolated from 77.7 and 70% of males and females, respectively, from 81.8% of children less than 5 years of age, and from 75.0% of patients of all age groups. The study indicates the important role of serotype 2b meningococci in causing the increased incidence of meningitis and further substantiates the important association of the serotype 2b determinant with group B serotype 2 meningococcal disease in Canada.

Bacterial Outer Membrane Proteins↗
PubMed2005

Post-PCV7 changes in colonizing pneumococcal serotypes in 16 Massachusetts communities, 2001 and 2004.

OBJECTIVE: The introduction of heptavalent conjugate pneumococcal vaccine (PCV7) has raised concerns for replacement with nonvaccine serotypes in both invasive disease and asymptomatic carriage. Analysis of colonizing serotypes among healthy children in the community provides critical data on such changes. METHODS: Nasopharyngeal specimens were obtained from children who were younger than 7 years during well-child or sick visits in primary care practices in 16 Massachusetts communities during 2001 and 2004. Susceptibility testing and serotyping were performed on isolated Streptococcus pneumoniae strains. Vaccination history with PCV7 was abstracted from the medical record. RESULTS: Among colonizing pneumococcal isolates, PCV7 serotypes decreased from 36% to 14%, and non-PCV7 serotypes increased from 34% to 55%. Overall carriage did not change (26% to 23%); neither did carriage of potentially cross-reactive serotypes (30% to 31%). The most common non-PCV7 serotypes were serotypes 11, 15, and 29. There was a substantial increase in penicillin nonsusceptibility from 8% to 25% in non-PCV7 serotypes; 35% were highly resistant to penicillin. Penicillin nonsusceptibility increased from 45% to 56% among PCV7 serotypes while remaining stable among PCV7 potentially cross-reactive strains (51% vs 54%). CONCLUSIONS: Pneumococcal colonization has changed after the introduction of PCV7, both in serotype distribution and in patterns of antibiotic resistance. The frequency of nonvaccine strains has increased, and the proportion of nonvaccine isolates that are not susceptible to penicillin has tripled. This shift toward increased carriage of nonvaccine serotypes warrants vigilance for changes in the epidemiology of invasive pneumococcal disease.

Child↗
PubMed1999

Antibody reactive with Porphyromonas gingivalis serotypes K1-6 in adult and generalized early-onset periodontitis.

BACKGROUND: Six serotypes of Porphyromonas gingivalis have recently been described. We sought to test the hypothesis that serotype specific carbohydrates from these strains are important antigens that elicit potent immune responses. METHODS: Serum concentrations of IgG reactive with P. gingivalis serotypes K1-K6 were determined for 28 adult (AP) and 28 generalized early-onset (G-EOP) periodontitis patients previously determined to be seropositive for a broken cell preparation of P. gingivalis. To confirm relationships suggested for K1, K2, and K6 in the analysis of initial data, the study population was increased to 133. RESULTS: Frequency of seropositivity for the 6 serotypes ranged from 26 to 54% of subjects. IgG concentrations ranged from 0 to 453 microg/ml with many subjects seropositive to more than one serotype. Concentrations for the subset of patients who was seropositive were high (mean responses ranged from 20 to 105 microg/ml for the 6 serotypes). Significant correlations between seropositivity to serotypes K1 and K5 as well as between K5 and K6 were found. CONCLUSIONS: We examined the relationship of diagnosis, race, gender, smoking, probing depth, attachment loss, and antibody reaction with the P. gingivalis serotypes by analysis of variance. Initial findings suggested potential relationships between diagnosis, smoking, race, gender, and antibody reactive with serotypes K1, K2, and K6. A significant relationship did exist between smoking and decreased antibody reactive with P. gingivalis serotype K2. No other relationships were substantiated. We also examined the IgG subclass distribution and found that responses were almost exclusively IgG2. These data support the concept that antibody responses to all 6 serotypes are common in both AP and G-EOP and that these K serotype carbohydrates elicit potent IgG2 responses.

Adult↗
PubMed1983

Antigenic variations of Candida albicans in vivo and in vitro--relationships between P antigens and serotypes.

Serum samples from patients with candidosis and from rabbits experimentally infected with serotype B C. albicans strains consistently showed higher antibody titers against Candida strains with serotype A antigens than strains with serotype B antigens, in indirect fluorescent antibody tests. When sera from rabbits infected with C. albicans serotype B strains were absorbed with blastospores of the homologous strain they continued to react against strains of C. albicans serotype A and a C. tropicalis strain with serotype A antigens. Serotype A-specific antisera reacted against tissue forms of C. albicans serotype B in vivo and against serotype B germ tubes, but not their parent blastospores, in vitro. These findings suggest that C. albicans B serotype cells may sometimes express, in vitro or in vivo, one or several antigens so far considered as specific of serotype A. The results have implications for the classical concept of C. albicans serotypes and for the serological diagnosis of candidosis in relation with the previously described strain P variable antigens.

Agglutination Tests↗
PubMed1982

Ureaplasma urealyticum serotypes isolated from cases of female sterility.

Ureaplasma strains isolated from vagina, cervix, endometrium or urine of 45 infertile women (121 strains) and 54 fertile women (145 strains) were tested using the growth inhibition test with standard Ureaplasma sera (types 1 through 8). Serotypes 3, 4 and 6 were more frequently recovered. Predominance of serotype 3 (35.5% of cases) over serotype 6 (22.2% of cases) in the infertile group, and conversely, predominance of serotype 6 (33.3%) over serotype 3 (20.3%) in the fertile women were noted. Serotype 4 was present in 15.5% of infertile subjects and 14.8% of the fertile ones. Serotypes 1, 2, 7 and 8 were found in proportions ranging between 1.8 and 11.1% of the positive Ureaplasma subjects. Serotypes 5 was never isolated. The predominant frequency of serotype 3 in infertile women and of serotype 6 in fertile women was observed regardless of the samples from which the strains were isolated. The proportion of non-typable strains amounted to 1.6% of strains isolated from the infertile group and to 5.5% of those isolated from the fertile group. The authors suggest the utilization of the serotyping scheme with 8 serotypes (Black and Shepard) for epidemiological studies including the transmissibility of genital Ureaplasma infection.

Bacteriuria↗
PubMed1996

New serotype candidate of Neisseria meningitidis.

In spite of a large collection of MAbs used for the whole-cell ELISA (WCE) in the National Reference Laboratory for Meningococcal Infections in Prague 50-80% of N.meningitidis strains isolated in the Czech Republic remained non-typable (NT) and/or non-subtypable (NST). A project focused on the problem of NT/NST N.meningitidis was started and the new serotype candidate designated "22" resulted from this research. This paper presents the method of preparing and testing of the monoclonal antibody (MAb) specific for this new serotype and the first experience acquired from using it. The new serotype-specific MAb is of IgG3 class, does not react with any serotype/subtype reference strains and reacts in WCE with the strain used for its production and with some other NT/NST strains in the dilution 1:1,000. A collection of 97 N.meningitidis B:NT strains isolated from cerebrospinal fluid and/or blood of patients with invasive disease in the Czech Republic since 1973 to 1995 was serotyped using the new serotype "22"-specific MAb and 37 of these strains (38.2%) gave positive WCE result. The total number of 59 N.meningitidis B:NT strains isolated in 1995 from various clinical situations were serotyped and 26 of them (44.1%) were positive with the new serotype "22"-specific MAb. Seven of these N.meningitidis B:NT strains isolated in 1995 from various clinical situations were serotyped and 26 of them (44.1%) were positive with the new serotype "22"-specific MAb. Seven of these N.meningitidis B: "22" strains were isolated from cerebrospinal fluid or blood of patients with invasive meningococcal disease and prevailed in the age group of 0-4 years (5 cases). The significance of the new serotype candidate was underlined recently, when this serotype "22" was recognized in N.meningitidis B strain isolated from a died 10 months old boy. These results indicate the epidemiological and clinical significance of the new serotype candidate "22" in the Czech Republic.

Animals↗
PubMed1997

Transmission and distribution of virus serotypes: African horse sickness in zebra.

The prevalence of African horse sickness (AHS) serotypes in zebra foals from the Kruger National Park, South Africa was examined for possible associations between serotypes and to estimate the basic reproduction number, R0. The distributions of serotypes between zebra were not independent in the 6- and 7-8-month-old age classes (P < 0.005). This does not necessarily imply biological interactions between serotypes, as heterogeneity in host-vector transmission rates can generate non-independent distributions of serotypes. Both age and month of capture were significant factors in the number of serotypes infecting each zebra (P < 0.0001). Pairwise, positive associations between non-cross-reacting serotypes were found in the 7-8-month-old class only. For AHS overall, estimates of R0 ranged from 31-68. Assuming serotypes are transmitted independently, estimates of R0 for individual serotypes ranged from 10 for serotype 1 to 23 for serotype 6. The wide range of estimates emphasizes the need for a better understanding of serotype transmission and interactions in AHS.

African Horse Sickness↗
PubMed2002

Biosensor for dengue virus detection: sensitive, rapid, and serotype specific.

A serotype-specific RNA biosensor was developed for the rapid detection of Dengue virus (serotypes 1-4) in blood samples. After RNA amplification, the biosensor allows the rapid detection of Dengue virus RNA in only 15 min. In addition, the biosensor is portable, inexpensive, and very easy to use, making it an ideal detection system for point-of-care and field applications. The biosensor is coupled to the isothermal nucleic acid sequence-based amplification (NASBA) technique with which small amounts of virus RNA are amplified using a simple water bath. During the NASBA reaction, a generic sequence is attached to all RNA molecules as described earlier (Wu, S. J.; Lee, E. M.; Putvatana, R.; Shurtliff, R. N.; Porter, K R.; Suharyono, W.; Watt, D. M.; King, C. C.; Murphy, G. S.; Hayes, C. G.; Romano, J. W. J. Clin. Microbiol. 2001, 39, 2794-2798.). It has been shown earlier that Dengue virus can be detected specifically using two DNA probes: a first probe hybridized with the attached generic sequence and, therefore, bound to every amplified RNA molecule; and a second probe either bound to all four Dengue virus serotypes or chosen to be specific for only one serotype. These probes were utilized in the biosensor described in this publication. For a generic Dengue virus biosensor, the second probe is complementary to a conserved region found in all Dengue serotypes. For identification of the individual Dengue virus serotypes, four serotype-specific probes were developed (Wu, S. J.; Lee, E. M.; Putvatana, R.; Shurtiff, R. N.; Porter, K. R.; Suharyono, W.; Watt, D. M.; King, C. C.; Murphy, G. S.; Hayes, C. G.; Romano, J. W. J. Clin. Microbiol. 2001, 39, 2794-2798.). The biosensor is a membrane-based DNA/RNA hybridization system using liposome amplification. The generic DNA probe (reporter probe) is coupled to the outside of dye-encapsulating liposomes. The conserved or Dengue serotype specific probes (capture probes) are immobilized on a polyethersulfone membrane strip. Liposomes are mixed with amplified target sequence and are then applied to the membrane. The mixture is allowed to migrate along the test strip, and the liposome-target sequence complexes are immobilized in the capture zone via hybridization of the capture probe with target sequence. The amount of liposomes present in the immobilized complex is directly proportional to the amount of target sequence present in the sample and can be quantified using a portable reflectometer. The different biosensor components have been optimized with respect to sensitivity and, foremost, specificity toward the different serotypes. An excellent correlation to a laboratory-based detection system was demonstrated. Finally, the assay was tested using a limited number of clinical human serum samples. Although Dengue serotypes 1, 2 and 4 were identified correctly, serotype 3 displayed low cross-reactivity with biosensors designed for detection of serotypes 1 and 4.

Animals↗