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PubMed1990

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

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 1,000- to 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.

Feces↗

PubMed1996

Reverse transcriptase-polymerase chain reaction for bcr/abl fusion in chronic myelogenous leukemia.

Reverse transcriptase-polymerase chain reaction (RT-PCR) has shown promise as a means of detecting low levels of cells bearing the Philadelphia chromosome (Ph1) and for detecting cytogenetically inapparent ("masked") Ph1 in patients with chronic myelogenous leukemia (CML). For detection by karyotyping, dividing cells must be used, precluding use of peripheral blood samples in cases with low peripheral blood blast counts. Reverse transcriptase-polymerase chain reaction was performed in 83 bone marrow and 30 peripheral blood samples from patients with CML to compare results with karyotyping and to evaluate utility of this test on peripheral blood samples. Using isolated total cellular RNA and a single primer pair, cDNA was transcribed, amplified, electrophoresed, and probed for bcr/abl fusion involving M-bcr exons 2 and 3 of the bcr gene. Fifty-three samples were from untreated or conventionally treated patients (pre-BMT), and 60 were from patients who had undergone bone marrow transplantation (post-BMT). Fifty of 53 pre-BMT samples were positive by RT-PCR. Two samples, negative by RT-PCR, had complex translocations, t(9;16;22) and t(4;14;22). One case was indeterminate by RT-PCR, but positive on retesting. Forty-five of 53 had Ph1 by karyotyping; 8 were negative, including 5 peripheral blood samples, 2 bone marrow samples with "masked" Ph1, and 1 bone marrow sample with poor growth. Thirty-five of 60 post-BMT samples were positive by RT-PCR. Fourteen of 60 post-BMT samples had Ph1 by karyotyping. Of the RT-PCR+/Ph1- cases, most showed a weak but definite band by RT-PCR, suggesting a low level of the bcr/abl fusion gene. Nineteen patients had concurrent peripheral blood and bone marrow samples analyzed by RT-PCR and karyotyping. Of 16 patients with satisfactory RNA extraction, 15 had concordant results by RT-PCR. Five patients had adequate metaphase cells for karyotypic analysis. All had Ph1 in bone marrow, but were negative in peripheral blood. Our results indicate that RT-PCR for detection of bcr/abl fusion is more sensitive than karyotyping in pre- and post-BMT samples. Furthermore, RT-PCR can be successfully performed on peripheral blood, yielding excellent correlation with bone marrow samples.

Base Sequence↗

PubMed2004

Cardiotropic viruses in the myocardium of children with end-stage heart disease.

BACKGROUND: Transplantation has become a lifesaving procedure for children with end-stage heart failure. The long-term outcome for children who undergo transplantation has been of considerable interest, but the causes of graft failure and death are largely unknown, and the role of pre-transplant viral infection is unclear. METHODS: Myocardial samples from 80 explanted hearts from children with end-stage heart disease caused by congenital heart disease (CHD), cardiomyopathy, or chronic rejection were analyzed using polymerase chain reaction and reverse-transcriptase polymerase chain reaction for cardiotropic viruses using virus-specific primers. We used immunohistochemical analysis of cytoskeletal proteins to evaluate myocyte architecture. RESULTS: We identified parvoviral genomes in 6 patients (3 with CHD and 3 with cardiomyopathy). We detected no other viruses. Immunohistochemistry showed normal staining for key components of the cytoskeleton/sarcolemma, sarcomere, and nuclear membrane in the 6 virus-positive samples. The clinical outcome of these children was worse (4 long-term survivors, but 2 deaths) than for individuals without the genome. CONCLUSIONS: Detecting viruses within the myocardium at the point of end-stage heart failure is not common, regardless of the primary pathology. However, the presence of viruses may result in poor outcome for the patient.

Child↗

PubMed2000

The detection of astrovirus in sludge biosolids using an integrated cell culture nested PCR technique.

The work presented here demonstrates the utility of the integrated cell culture-reverse transcriptase-polymerase chain reaction (ICC-RT-PCR) coupled with nested PCR to detect human astroviruses and enteroviruses in sludge biosolids. Viruses were concentrated by beef extract elution and organic flocculation prior to analysis by a plaque assay and ICC-RT-PCR. Astroviruses were detected in all but one sample and all of the samples were positive for enteroviruses. We have demonstrated the prevalence and frequency ofastrovirus in sludge and validated the ICC-RT-PCR/nested PCR technique as a useful tool to detect viruses in sludge.

Caco-2 Cells↗

PubMed2000

The AROM gene, spliced mRNAs encoding new DNA/RNA-binding proteins are transcribed from the opposite strand of the melanin-concentrating hormone gene in mammals.

Melanin-concentrating hormone (MCH) mRNA expression is induced by nerve growth factor and lithium in PC12 cells, whereas three large MCH RNA species are found in untreated cells. In this study, we investigated the structures, regulations of expression, and putative functions of these transcripts. Northern blot, rapid amplification of cDNA ends-polymerase chain reaction, reverse transcriptase-polymerase chain reaction, and sequencing experiments demonstrated that they are antisense RNAs complementary to the MCH gene. Two classes of antisense RNAs could be discriminated as follows: 1) non-coding unspliced RNAs that overlap mainly the coding part of the MCH gene; 2) spliced variant mRNAs complementary to the 3'-flanking end of the MCH gene and that encode putative proteins containing DNA/RNA binding domains. We named this new transcriptional unit AROM for antisense-RNA-overlapping-MCH gene. Spliced variant AROM mRNAs are expressed in a broad range of rat organs. Western blot and immunohistochemistry experiments revealed several proteins with cytoplasmic but also nuclear localization in PC12 cells. Time course studies during nerve growth factor and lithium treatment of PC12 cells indicated a reciprocal regulation of the MCH and AROM gene transcripts, reflected also at the level of AROM proteins. The major translational product is a 64-kDa protein (AROM-p64). Recombinant AROM-p64 displayed high binding to single-stranded DNA and poly(A) homopolymers suggesting that this protein could play a role in mRNA maturation/metabolism.

Amino Acid Sequence↗

PubMed2001

The phenylalanine ammonia-lyase gene family in raspberry. Structure, expression, and evolution.

In raspberry (Rubus idaeus), development of fruit color and flavor are critically dependent on products of the phenylpropanoid pathway. To determine how these metabolic functions are integrated with the fruit ripening program, we are examining the properties and expression of key genes in the pathway. Here, we report that L- phenylalanine ammonia-lyase (PAL) is encoded in raspberry by a family of two genes (RiPAL1 and RiPAL2). RiPAL1 shares 88% amino acid sequence similarity to RiPAL2, but phylogenetic analysis places RiPAL1 and RiPAL2 in different clusters within the plant PAL gene family. The spatial and temporal expression patterns of the two genes were investigated in various vegetative and floral tissues using the reverse transcriptase competitor polymerase chain reaction assay. Although expression of both genes was detected in all tissues examined, RiPAL1 was associated with early fruit ripening events, whereas expression of RiPAL2 correlated more with later stages of flower and fruit development. Determination of the absolute levels of the two transcripts in various tissues showed that RiPAL1 transcripts were 3- to 10-fold more abundant than those of RiPAL2 in leaves, shoots, roots, young fruits, and ripe fruits. The two RiPAL genes therefore appear to be controlled by different regulatory mechanisms.

Acyltransferases↗

PubMed2005

Aetiological role of viral and bacterial infections in acute adult lower respiratory tract infection (LRTI) in primary care.

BACKGROUND: Lower respiratory tract infections (LRTI) are a common reason for consulting general practitioners (GPs). In most cases the aetiology is unknown, yet most result in an antibiotic prescription. The aetiology of LRTI was investigated in a prospective controlled study. METHODS: Eighty adults presenting to GPs with acute LRTI were recruited together with 49 controls over 12 months. Throat swabs, nasal aspirates (patients and controls), and sputum (patients) were obtained and polymerase chain reaction (PCR) and reverse transcriptase polymerase chain reaction (RT-PCR) assays were used to detect Streptococcus pneumoniae, Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, influenza viruses (AH1, AH3 and B), parainfluenza viruses 1-3, coronaviruses, respiratory syncytial virus, adenoviruses, rhinoviruses, and enteroviruses. Standard sputum bacteriology was also performed. Outcome was recorded at a follow up visit. RESULTS: Potential pathogens were identified in 55 patients with LRTI (69%) and seven controls (14%; p<0.0001). The identification rate was 63% (viruses) and 26% (bacteria) for patients and 12% (p<0.0001) and 6% (p = 0.013), respectively, for controls. The most common organisms identified in the patients were rhinoviruses (33%), influenza viruses (24%), and Streptococcus pneumoniae (19%) compared with 2% (p<0.001), 6% (p = 0.013), and 4% (p = 0.034), respectively, in controls. Multiple pathogens were identified in 18 of the 80 LRTI patients (22.5%) and in two of the 49 controls (4%; p = 0.011). Atypical organisms were rarely identified. Cases with bacterial aetiology were clinically indistinguishable from those with viral aetiology. CONCLUSION: Patients presenting to GPs with acute adult LRTI predominantly have a viral illness which is most commonly caused by rhinoviruses and influenza viruses.

Acute Disease↗

PubMed2001

Adult human heart microvascular endothelial cells are permissive for non-lytic infection by human cytomegalovirus.

OBJECTIVE: Human cytomegalovirus (CMV) infection has been linked to chronic heart disease. The mechanism of CMV dissemination to the heart remains unknown. CMV antigens and nucleic acid sequences have been detected in endothelial cells (ECs) in vivo, and ECs are fully permissive hosts to CMV replication in vitro. This report examines the characteristics of CMV replication in primary cultures of human heart microvascular ECs (HHMECs). METHODS: Capillary ECs were isolated from heart tissue biopsies of six patients at the time of heart surgery. HHMECs were infected with CMV and viral antigens were detected by immunofluorescence assay using monoclonal antibodies as specific reagents. Cytokine and chemokine release in the supernatant of sham- and CMV-infected cells was quantitated by ELISA. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to analyse expression of mRNA for adhesion molecules. RESULTS: CMV was found to productively infect HHMECs without cytolytic effects. Infected cultures released high levels of pro-inflammatory chemokines and enhanced their adhesion molecule expression. CONCLUSIONS: Our data provide new insights into the mechanism of CMV dissemination to the heart, signalling the need for further investigation of the pathogenetic role of this virus in cardiac disorders.

Adult↗

PubMed2006

Heterogeneous phenotype in a family with compound heterozygous parkin gene mutations.

BACKGROUND: Mutations in the parkin gene (PRKN) cause autosomal recessive early-onset Parkinson disease (EOPD). OBJECTIVE: To investigate the presence of mutations in the PRKN gene in a white family with EOPD and the genotype-phenotype correlations. DESIGN: Twenty members belonging to 3 generations of the EOPD family with 4 affected subjects underwent genetic analysis. Direct genomic DNA sequencing, semiquantitative polymerase chain reaction, real-time quantitative polymerase chain reaction, and reverse-transcriptase polymerase chain reaction analyses were performed to identify the PRKN mutation. RESULTS: Compound heterozygous mutations (T240M and EX 5_6 del) in the PRKN gene were identified in 4 patients with early onset (at ages 30-38 years). Although heterozygous T240M and homozygous EX 5_6 del mutations in the PRKN gene have been previously described, this is, to our knowledge, the first report of these mutations in compound heterozygotes. The phenotype of patients was that of classic autosomal recessive EOPD characterized by beneficial response to levodopa, relatively slow progression, and motor complications. All heterozygous mutation carriers (T240M or EX 5_6 del) and a 56-year-old woman who was a compound heterozygous mutation carrier (T240M and EX 5_6 del) were free of any neurological symptoms. CONCLUSIONS: Compound heterozygous mutations (T240M and EX 5_6 del) in the PRKN gene were found to cause autosomal recessive EOPD in 4 members of a large white family. One additional member with the same mutation, who is more than 10 years older than the mean age at onset of the 4 affected individuals, had no clinical manifestation of the disease. This incomplete penetrance has implications for genetic counseling, and it suggests that complex gene-environment interactions may play a role in the pathogenesis of PRKN EOPD.

Adult↗

PubMed2003

Up-regulated humoral immune response in the soft tick, Ornithodoros moubata (Acari: Argasidae).

Ticks have an efficient defense system for preventing microbial infection. The antimicrobial peptide defensin is one effective molecule in this system. Here we investigated immune competence and the involvement of defensin in the humoral defense of the soft tick, Ornithodoros moubata. Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) revealed that gene expression of all four defensin isoforms was up-regulated by bacteria or bacterial components. Defensin gene up-regulation by hemocoelic inoculation of bacteria involves the midgut and granulocytes. In immunodetection analysis, immunization by bacterial injection increases the relative concentration of defensin-like material in the hemolymph plasma. Furthermore, elevated antibacterial activity against Gram-positive bacteria but not against Gram-negative bacteria was observed after immunization by a liquid growth inhibition assay. Therefore, enhanced anti-Gram-positive bacterial activity appears to be partially dependent on the release of defensin into the hemolymph. These findings demonstrate that defensin plays an important role in the up-regulated humoral response of O. moubata.

Animals↗

PubMed2005

Expression patterns of S100A7 (psoriasin) and S100A9 (calgranulin-B) in keratinocyte differentiation.

S100 proteins are involved in many biological processes. S100A7 and S100A9 have been shown to be markedly upregulated both in ductal carcinoma in situ of the breast and in psoriasis. We have examined the relationship between keratinocyte differentiation and the expression of the two proteins. Using Western blot analysis and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), both S100A7 and S100A9 were shown to be induced in normal primary keratinocytes (HEKn), when differentiation was promoted by high extracellular calcium, loss of contact with extracellular matrix and confluent conditions, as previously reported for S100A7 in mammary epithelial cells. Differentiation was confirmed by using RT-PCR for the differentiation marker keratin-1. Using immunohistochemistry with monoclonal antibodies, we compared the expression of the two proteins in a spectrum of conditions of dysregulated keratinocyte differentiation. We found a strikingly similar distribution of the proteins. Their expression correlated with the degree of keratinocyte differentiation. They were both absent in undifferentiated basalioma and strongly expressed in carcinoma in situ, as well as in keratoacanthoma and differentiated squamous cell carcinoma. In normal epithelium, they were expressed in the superficial, differentiated region of the epithelium rather than in the basal region. These findings support the hypothesis that these two S100 proteins are involved in keratinocyte differentiation.

Blotting, Western↗

PubMed1995

Mechanism of androgen-induced thymolysis in rats.

To investigate the mechanism of androgen-induced thymolysis, the effects of various androgens, including testosterone (T), 19-nortestosterone, and 7 alpha-methyl-19-nortestosterone (MENT), were compared with those of estradiol and dexamethasone (DEX) in intact, castrated, and adrenalectomized male rats. The potency comparisons on thymus regression, based on mass of steroids, showed DEX to be the most potent, followed by estradiol and the androgens. Among the androgens, MENT was the most potent, followed by nortestosterone and T, an order similar to their anabolic potency on muscle. As the thymolytic effects of T and MENT were not altered by the concomitant administration of an aromatase inhibitor or a 5-reductase inhibitor, it was concluded that the effects of androgens were not mediated by their conversion to estrogens or 5 alpha-reduced metabolites. Involvement of glucocorticoid receptors in androgen action was excluded because mifepristone (an antiglucocorticoid) blocked DEX-induced, but not T- or MENT-induced, thymus regression. Flutamide, an antiandrogen, significantly blocked the thymolytic effect of T and MENT, providing further support for this conclusion. This suggested that the thymolytic action of androgens is an intrinsic property mediated via androgen receptors (AR). The occurrence of AR in the thymus was demonstrated by binding assays and the presence of AR messenger RNA (mRNA) by reverse transcriptase-polymerase chain reaction. Quantitative reverse transcriptase-polymerase chain reaction for AR mRNA in the thymus showed 6-fold more AR mRNA in the thymic epithelial cells than in the thymocytes. However, epithelial cells represent only a small fraction of the thymus. Hence, it is hypothesized that the androgens produce their thymolytic effects by stimulating the secretion of a factor(s) by the thymic epithelial cells that, in turn, causes regression of the thymus.

Adrenalectomy↗

PubMed2001

Activation of HIV in human skin by ultraviolet B radiation and its inhibition by NFkappaB blocking agents.

To determine whether ultraviolet B (UVB) irradiation leads to activation of HIV in human skin, we conducted prospective and controlled studies in two academic medical centers in Texas from July 1995 to April 1999. HIV-positive patients with UV-treatable skin diseases were enrolled at each center, 18 subjects at one and 16 at the other. In one center, specimens from lesional and nonlesional skin biopsies were taken before and after sham- or UVB-irradiation administered in vivo or in vitro. In the other center, UVB phototherapy was administered three times weekly and specimens from skin biopsies were taken before and after 2 weeks (six treatments). Cutaneous HIV load was assessed using reverse transcriptase-polymerase chain reaction and reverse transcriptase-polymerase chain reaction in situ hybridization. UVB irradiation led to a 6-10-fold increase in the number of HIV in skin. To ascertain a role for nuclear factor kappa B (NFkappaB) in UVB-inducible HIV activation, two types of blockers, NFkappaB oligonucleotide decoy and sodium salicylate, were tested; each inhibited UVB-inducible HIV activation in skin partially. We conclude that UVB irradiation leads to increased numbers of HIV in human skin via processes that include release of cytoplasmic NFkappaB.

HIV↗

PubMedDate not supplied

Comparison of virus isolation, immunohistochemistry, and reverse transcriptase-polymerase chain reaction procedures for detection of turkey coronavirus.

A reverse transcriptase-polymerase chain reaction (RT-PCR) procedure and two monoclonal antibody (MAb)-based immunohistochemical procedures were developed for detection of turkey coronavirus (TCV) in tissues and intestinal contents/dropping samples. The RT-PCR, MAb-based fluorescent antibody (FA), and MAb-based immunoperoxidase (IP) procedures were compared with virus isolation (VI) for detection of TCV in experimentally infected turkeys. TCV was detected in experimentally infected turkeys as early as day 1 postexposure (PE) by each of the four detection procedures. TCV was detected as late as day 35 PE by FA or IP and days 42 and 49 PE by VI and RT-PCR, respectively. With VI as a reference, sensitivity and specificity of RT-PCR were 93% and 92%, respectively; specificity of both FA and IP was 96%, and sensitivities were 69% and 61%, respectively. Each of the examined procedures was highly specific, but the RT-PCR procedure was also highly sensitive. These findings demonstrate the utility of both immunohistochemistry and RT-PCR for detection of TCV. In addition, the findings indicate that RT-PCR is a highly sensitive and specific alternative to conventional diagnostic procedures.

Animals↗

PubMed2005

Molecular detection and serotypic characterization of dengue viruses by single-tube multiplex reverse transcriptase-polymerase chain reaction.

This study was designed to develop a reverse transcriptase-polymerase chain reaction (RT-PCR)-based rapid and cost-effective diagnostic test for detection as well as serotypic characterization of dengue viruses in the acute phase of illness. In this regard, 2 methods of RT-PCR, namely, 2-step nested RT-PCR and 1-tube multiplex RT-PCR, have been evaluated. One-tube multiplex RT-PCR was found superior; thus, its sensitivity and specificity were compared with gold standard "cell culture". In the present study, serum samples of all individuals were evaluated by cell culture and RT-PCR. Of 200 clinically suspected patients tested, 66 were found to be positive for the dengue virus RNA and could be successfully characterized into the serotypes. Of these 66, 62 patients were found to be serologically positive by Dengue Duo IgM and IgG Rapid Strip test (Pan Bio, Windsor, Australia). The sensitivity of the cell culture procedure was found to be lower; 63% as compared with multiplex RT-PCR, with predictive value of positive test being 100% and predictive value of negative test being 84.8%. Specificity was found to be 100% for both the assays. It is thus proposed that 1-tube multiplex RT-PCR-based assay would prove to be an extremely useful tool for routine laboratory diagnosis.

Aedes↗

PubMed1997

Identification of cytokine and adhesion molecule mRNA in murine lung tissue and isolated T cells and eosinophils by semi-quantitative reverse transcriptase-polymerase chain reaction.

We have used a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay to detect the expression of mRNA for inflammatory cytokines, integrins and selectins in murine lung tissue, and T cells and eosinophils isolated from lung and bronchoalveolar lavage (BAL) fluid in an in vivo model of ovalbumin (OA)-induced airway inflammation. RNA was isolated from whole lung tissue at 1, 6, 24, 48, 72 h, and 7 days after OA inhalation. mRNA for the Th2 cytokines, IL-4, -5, -6, -10 and -13 in OA-sensitized mice were significantly elevated compared with non-sensitized mice. IL-2, TNF-beta, and eotaxin mRNA were also increased, but IFN-gamma mRNA was not. P- and E-selectin mRNA levels were also enhanced in lung tissue between 6 and 72 h after challenge. Lung T cells were isolated by cell sorting with a flow cytometer at 3, 12, 24, 48 and 72 h after challenge. mRNA levels for IL-5 and -10 were greater in T cells from OA-sensitized and -challenged mice than controls at 24 h. BAL fluid from OA-sensitized and -challenged mice also had significantly higher IL-5 levels than controls. BAL fluid T cells and eosinophils were obtained at 48 and 72 h after aerosol challenge and were purified by cell sorting. Messenger RNA for IL-1 alpha, -2, -4, -5, -10, IFN-gamma, and beta 1 were detected in T cells at both time points. Transcripts for IL-1 alpha, -4, -5, -13, TNF-alpha and beta, and alpha 4, beta 1 and beta 7 were also identified in BAL eosinophils. These data show that in addition to murine lung T cells, airway eosinophils may also contribute to the inflammatory response by their ability to express mRNA for cytokines and integrins.

Animals↗

PubMed1997

Rapid diagnosis of dengue viremia by reverse transcriptase-polymerase chain reaction using 3'-noncoding region universal primers.

A reverse transcriptase-polymerase chain reaction (RT-PCR) method was developed as a rapid diagnostic test of dengue viremia. To detect dengue viruses in serum or plasma specimens, a pair of universal primers was designed for use in the RT-PCR. Using these primers, the 3'-noncoding region of dengue virus types 1, 2, 3, and 4 could be amplified, but not those of other flaviviruses, such as West Nile virus, Japanese encephalitis virus, and yellow fever virus, or the alphavirus Sindbis virus. The sensitivity of the RT-PCR assay was similar to that of a quantitative fluorescent focus assay of dengue viruses in cell culture. Combining a silica method for RNA isolation and RT-PCR dengue virus could be detected in a 6-hr assay. In a preliminary study using this method, we detected dengue virus in 38 of 39 plasma specimens from which dengue virus had been isolated by mosquito inoculation. We then applied this method for detecting dengue viremia to 117 plasma samples from 62 children with acute febrile illnesses in a dengue-endemic area. We detected dengue viremia in 19 of 20 samples obtained on the day of presentation, which had been confirmed as acute dengue infection by mosquito inoculation and antibody responses. The overall sensitivity of this method was 91.4% (32 of 35; 95% confidence interval [CI] = 82.2-100%). The results from testing plasma samples from febrile nondengue patients showed a specificity of 95.4% (42 of 44; 95% CI = 89.3-100%).

Acute Disease↗

PubMed2001

Application of reverse transcriptase-polymerase chain reaction for detection of Rift Valley Fever viral antigen from mosquito.

A reverse transcriptase polymerase chain reaction (RT-PCR) was applied to detect Rift Valley Fever Virus (RVFV) in Culex pipiens mosquito pools collected from Alexandria and Behira governorates of Egypt (50 pools each). All mosquito pools were subjected to double sandwich enzyme linked immunosorbent assay (ELISA) technique to detect RVF viral antigen. Out of all 100 mosquito pools, only 18(18%) were positive by ELISA, 10(20%) out of 50 pools were positive in Behira governorate and 8(16%) were positive in Alexandria governorate. All positive samples (18) in addition to two negative samples (one was used as a negative control and the other was used as a positive control after addition of 1.0 ml. of 103 inactivated RVF virus) were subjected to RT-PCR. Out of these 18 positive samples by ELISA, only 7(38.89%) were positive for RVF Virus by RT-PCR. These results gave the possibilities of existence of other phleboviruses that cross react with RVF Virus.

Animals↗