Real-time Real-time Reverse Transcription-polymerase Chain Reaction Research Articles

Newly Emerging Mutations in the Matrix Genes of the Human Influenza A(H1N1)pdm09 and A(H3N2) Viruses Reduce the Detection Sensitivity of Real-Time Reverse Transcription-PCR

New variants of the influenza A(H1N1)pdm09 and A(H3N2) viruses were detected in Taiwan between 2012 and 2013. Some of these variants were not detected in clinical specimens using a common real-time reverse transcription-PCR (RT-PCR) assay that targeted the conserved regions of the viral matrix (M) genes. An analysis of the M gene sequences of the new variants revealed that several newly emerging mutations were located in the regions where the primers or probes of the real-time RT-PCR assay bind; these included three mutations (G225A, T228C, and G238A) in the A(H1N1)pdm09 virus, as well as one mutation (C163T) in the A(H3N2) virus. These accumulated mismatch mutations, together with the previously identified C154T mutation of the A(H1N1)pdm09 virus and the C153T and G189T mutations of the A(H3N2) virus, result in a reduced detection sensitivity for the real-time RT-PCR assay. To overcome the loss of assay sensitivity due to mismatch mutations, we established a real-time RT-PCR assay using degenerate nucleotide bases in both the primers and probe and successfully increased the sensitivity of the assay to detect circulating variants of the human influenza A viruses. Our observations highlight the importance of the simultaneous use of different gene-targeting real-time RT-PCR assays for the clinical diagnosis of influenza.

Evaluation of a new real-time reverse transcription polymerase chain reaction assay for detection of norovirus in fecal specimens

A new real-time reverse transcription polymerase chain reaction (RT-PCR) assay, the AccuPower Norovirus Real-time RT-PCR Kit, was evaluated in detection of human norovirus in stool specimens. Studies for detection limit, dynamic range, reproducibility, and cross-reactivity were performed. A total of 281 fecal specimens were tested using the AccuPower Norovirus Real-time RT-PCR Kit, and the results were compared with those obtained using another real-time RT-PCR system. Norovirus positivity and genotype were confirmed by direct sequencing. The lowest mean numbers of genome copies of GI and GII that could be detected by the assay were 12.3 and 5.6 RNA copies/reaction, respectively. The positive, negative, and overall percent agreements between the 2 real-time PCR assays were 99.0% (96/97), 95.1% (175/184), and 96.4% (271/281), respectively. The AccuPower Norovirus Real-time RT-PCR system showed good analytical and clinical performance and may be a useful diagnostic tool for norovirus infection.

Comparison of virus shedding after lived attenuated and pentavalent reassortant rotavirus vaccine

Transmission of rotavirus vaccine or vaccine-reassortant strains to unvaccinated contacts has been reported. Therefore, it is essential to evaluate and characterize the nature of vaccine–virus shedding among rotavirus vaccine recipients. Two groups of healthy infants who received a complete course of RotaTeq (RV5) or Rotarix (RV2) were enrolled (between March 2010 and June 2011) to compare fecal shedding for one month after each vaccine dose. Shedding was assessed using both enzyme immunoassay (EIA) and real-time reverse transcription-polymerase chain reaction (RT-PCR). Eighty-seven infants (34 girls and 53 boys) were enrolled in the study. After the first vaccine dose, the peak time of virus shedding occurred between day 4 and day 7, with positive detection rates of 80–90% by real-time RT-PCR and 20–30% by EIA. In both groups, vaccine shedding occurred as early as one day and as late as 25–28 days. Mixed effects logistic regression analysis of real-time RT-PCR data showed no significant differences between two groups when shedding rates were compared after the first vaccine dose (odds ratio [OR] 1.26; P=0.71) or after the second vaccine dose (odds ratio [OR] 1.26; P=0.99). However, infants receiving RV2 shed significantly higher viral loads than those receiving RV5 when compared after the first vaccine dose (P=0.001) and after the second dose (P=0.039). In terms of shedding rates detected by real-time RT-PCR, vaccine uptake of RV5 or RV2 among infants in Taiwan was comparable. Clinical significance of higher shedding viral loads in RV2 should be further observed.

Optimization of one-step real-time reverse transcription-polymerase chain reaction assays for norovirus detection and molecular epidemiology of noroviruses in Thailand

Noroviruses (NoVs) are an important human pathogen associated with acute viral gastroenteritis worldwide. NoVs display a significant amount of genetic heterogeneity, making it difficult to develop comprehensive detection assays. In this study, primer sets and probes were designed for a TaqMan®-based real-time reverse transcription-polymerase chain reaction (RT-PCR) for norovirus detection purposes. The assay was optimized and utilized as a multiplex real-time RT-PCR assay for genogroup I (GI) detection, and a singleplex real-time RT-PCR assay for genogroup II (GII) detection. The assays showed high specificity for NoV detection and no cross-reactivity was observed between GI and GII. The detection limit of the assay was as low as 10 and 50 RNA copies per reaction for GI and GII, respectively. The optimized protocol was employed to assess the presence of NoV strains in clinical samples collected throughout Thailand during December 2005 to November 2006. The percentage of NoV infections among children with acute gastroenteritis (case) was 23.8% (119/500) and for children without acute gastroenteritis (control) it was 6.8% (30/441). The frequency of NoV infections varied geographically, with the highest frequency observed in the central region and the lowest frequency in the northern region (P>0.0001). Of the 149 positive case and control specimens, GII was found to be the predominant genogroup (98.6%). Partial capsid sequences were successfully obtained from 67 NoV-positive specimens and a phylogenetic analysis was performed to genotype the viral strains. GII.4 was the most common genotype detected.

Temporospatial expression and cellular localization of VGLUT3 in the rat cochlea

Vesicular glutamate transporter 3 (VGLUT3) plays an important role in hearing, and VGLUT3 knockout mice are deaf. However, the mechanisms whereby VGLUT3 exerts its effects in the cochlea are not well established. Elucidating the developmental and aging dynamics of VGLUT3 localization and expression in the cochlea would aid a functional understanding of auditory glutamatergic transmission. In this study, we characterized the expression of VGLUT3 in rat auditory epithelium and spiral ganglion cells (SGCs) during postnatal development and with age using immunohistochemistry and quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). We observed VGLUT3 expression in cochlea inner hair cells (IHCs) and SGCs as early as postnatally, and its expression increased gradually with maturity. However, the expression of VGLUT3 in the IHCs decreased in the aging rats compared to postnatal day 60 (P60), and VGLUT3 was still expressed in the remaining SGCs in the aging rats. VGLUT3-immunopositive punctate structures were only found in the cytoplasm of SGCs, not IHCs, at P7, or later. Variation in VGLUT3 mRNA expression evaluated by quantitative real time RT-PCR was consistent with the immunohistochemical data except for no statistical difference between P60 and aging rats in the whole cochlea level. Our data suggest that VGLUT3 likely has developmental and physiological roles in the rat cochlea during postnatal development as well as later in life. VGLUT3 may have additional unknown roles in SGCs besides glutamate transport.

Anti-miRNA-23a Oligonucleotide Suppresses Glioma Cells Growth by Targeting Apoptotic Protease Activating Factor-1

Abnormal expression of microRNAs (miRNAs) is closely related to glioma, which is one of the most common malignant brain tumors. The current study is to identify the key miRNAs involved in the pathogenesis of glioma and to discover novel therapeutic targets for this disease. Total RNA was extracted from glioma tissues of 100 patients. The microRNA microarray and the northern blot were used to detect the changes of miRNAs expression in 7 pairs of glioma specimens. Relative expressions of miR-23a were validated by real-time reverse transcription polymerase chain reaction (RT-PCR) with specific Taqman probes. In order to evaluate the role of miR-23a, the miR-23a mimics and anti-miR-23a oligonucleotides were transfected to glioma cell lines; the cell proliferation, apoptosis, cell cycle percentage, cell migration and invasion abilities were evaluated in vitro. The target genes of miR-23a were also investigated using the bioinformatics tools. The expression of the apoptotic protease activating factor-1 (APAF1), which might be one of the direct targets of miR-23a, was also analyzed using the luciferase reporter assay and western blot analysis in 293T cells and glioma cell line, respectively. The microRNA microarray and the northern blot results showed that the expressions of miR-23a in glioma tissues were significantly upregulated. The miR-23a expression levels identified using real time RT-PCR in tumor tissues of 79 samples were higher than in the matched adjacent tissues. By transfection of anti-miR-23a oligonucleotide, the results showed that the proliferation, migration, and invasion of glioma cell lines were significantly suppressed. The bioinformatics searching results showed that APAF1 might be a direct target gene of miR-23a, and it was supported by the luciferase reporter gene assay and western blot analysis results. Finally, experiments showed that overexpression of APAF1 suppressed glioma cell growth and promoted cell apoptosis. Our findings characterized the expression properties of miR-23a, contributed to the function and molecular mechanism of miR-23a in glioma and implied that miR-23a might be employed as novel prognostic markers and therapeutic targets of glioma.

Development and Validation of One-Step SYBR Green Real-Time RT-PCR for the Rapid Detection of Newly Emerged Duck Tembusu Virus

Duck Tembusu virus (DTMUV) is a single-stranded positive-sense RNA virus that causes disease to emerge in duck flocks and results in huge economic losses to the duck industry. However, no vaccines and control measures are available in China to date. Development of reliable and fast detection methods is necessary to prevent and control this disease. Therefore, a one-step SYBR Green real-time reverse transcription polymerase chain reaction (RT-PCR) method is established here for DTMUV detection. The results show that the method can specifically detect DTMUV without cross-reactions with selected avian pathogens. The sensitivity of the assay was 1000 times greater than that of a conventional RT-PCR and able to test as few as 20 copies from RNA standard samples. The coefficients of variations of inter- and intra-assay values ranged from 0.09% to 0.36% and 0.1% to 0.23%, respectively. Testing 168 field samples and 96 experimentally infected samples by conventional RT-PCR and the one-step SYBR Green real-time RT-PCR, the positive rates were 35.1% and 73.8% from field samples and 30.2% and 64.6% from infected samples. The one-step SYBR Green real-time RT-PCR developed in this study was shown to be a sensitive, specific, high-throughput, cost-effective, and simple diagnostic tool for the rapid detection and epidemiological surveillance of the emerging DTMUV infection.

Wnt Inhibitory Factor 1 Deficiency Uncouples Cartilage and Bone Destruction in Tumor Necrosis Factor α–Mediated Experimental Arthritis

Wnt signaling plays a pivotal role in skeletal development and in the control of cartilage and bone turnover. We have recently shown that the secreted Wnt antagonist Wnt inhibitory factor 1 (WIF-1) is mainly expressed in the upper layers of epiphyseal and articular cartilage and, to a lesser extent, in bone. Nevertheless, WIF-1(-/-) mice develop normally. In light of these findings, we undertook this study to analyze the role of WIF-1 in arthritis. Expression analyses for WIF-1 were performed by real-time reverse transcription-polymerase chain reaction (RT-PCR). WIF-1(-/-) and tumor necrosis factor (TNF)-transgenic mice were crossbred, and the progression of arthritis in TNF-transgenic WIF-1(-/-) mice and littermate controls was evaluated. Structural joint damage was analyzed by histologic staining, histomorphometry, and micro-computed tomography. Wnt/β-catenin signaling was investigated by real-time RT-PCR and immunofluorescence on primary chondrocytes. WIF-1 expression was repressed by TNFα in chondrocytes and osteoblasts and down-regulated in experimental arthritis and in articular cartilage from patients with rheumatoid arthritis. WIF-1 deficiency partially protected TNF-transgenic mice against bone erosion and loss of trabecular bone, probably as a result of less osteoclast activity. In contrast, arthritis-related cartilage damage was aggravated by WIF-1 deficiency, while overexpression of WIF-1 attenuated cartilage degradation in TNF-transgenic mice. In chondrocytes, TNFα stimulated canonical Wnt signaling, which could be blocked by WIF-1, indicating a direct effect of TNFα and WIF-1 on Wnt signaling in this system. These data suggest that WIF-1 may take part in the fine-tuning of cartilage and bone turnover, promoting the balance of cartilage versus bone anabolism.

Environmental detection of genogroup I, II, and IV noroviruses by using a generic real-time reverse transcription-PCR assay.

Norovirus is the most common agent implicated in food-borne outbreaks and is frequently detected in environmental samples. These viruses are highly diverse, and three genogroups (genogroup I [GI], GII, and GIV) infect humans. Being noncultivable viruses, real-time reverse transcription-PCR (RT-PCR) is the only sensitive method available for their detection in food or environmental samples. Selection of consensus sequences for the design of sensitive assays has been challenging due to sequence diversity and has led to the development of specific real-time RT-PCR assays for each genogroup. Thus, sample screening can require several replicates for amplification of each genogroup (without considering positive and negative controls or standard curves). This study reports the development of a generic assay that detects all three human norovirus genogroups on a qualitative basis using a one-step real-time RT-PCR assay. The generic assay achieved good specificity and sensitivity for all three genogroups, detected separately or in combination. At variance with multiplex assays, the choice of the same fluorescent dye for all three probes specific to each genogroup allows the levels of fluorescence to be added and may increase assay sensitivity when multiple strains from different genogroups are present. When it was applied to sewage sample extracts, this generic assay successfully detected norovirus in all samples found to be positive by the genogroup-specific RT-PCRs. The generic assay also identified all norovirus-positive samples among 157 archived nucleic acid shellfish extracts, including samples contaminated by all three genogroups.

Usutu Virus in Migratory Song Thrushes, Spain

Usutu Virus in Migratory Song Thrushes, Spain

Estrogen and estrogen receptor induce matrix metalloproteinase-26 expression in endometrial carcinoma cells

The human matrix metalloproteinase (MMP)-26, also called matrilysin-2 or endometase, has been isolated as a matrilysin (MMP-7) homolog. Several reports describe that MMP-26 may be related to the development of endometrial carcinomas. Total RNAs were isolated from 51 normal endometrial tissue samples, 6 endometrial hyperplasia tissue samples and 30 endometrial carcinomas. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed to evaluate MMP-26 mRNA expression levels. We examined the effect of estrogen and its receptor (ER) on MMP-26 expression in endometrial carcinoma cell lines by real-time RT-PCR, western blot analysis and luciferase assays. To examine protein-DNA binding between ER and MMP-26 promoter, we performed chromatin immunoprecipitation (ChIP) assay. Real-time RT-PCR analysis revealed that MMP-26 mRNA expression was significantly higher in the normal human endometria and hyperplasias compared with that in endometrial carcinomas. Estrogen not only transactivated the MMP-26 promoter activity but also enhanced endogenous MMP-26 expression. The MMP-26 promoter region contains a putative ER response element (ERE). Nuclear ER protein interacted with ERE on the MMP-26 promoter by ChIP assay. We found a significant difference in MMP-26 expression in normal and malignant endometrial tissue samples and that estrogen induced MMP-26 expression. Estrogen may induce endometrial hyperplasia but not endometrial carcinoma. Our results provide evidence that regulation of MMP-26 promoter activity by estrogen may represent a mechanism for endometrial carcinogenesis.

Vasohibin-1 Expression Is Regulated by Transforming Growth Factor-β/Bone Morphogenic Protein Signaling Pathway Between Tumor-Associated Macrophages and Pancreatic Cancer Cells

Vasohibin-1 has been detected in endothelial cells as an intrinsic angiogenesis inhibitor. Both tumor-associated macrophages (TAMs) and transforming growth factor-β (TGF-β)/bone morphogenic protein (BMP) signaling have been reported to promote angiogenesis in cancer. However, whether vasohibin-1 expression is regulated by TGF-β/BMP signaling between TAMs and cancer cells remains unclear. The expression of TGF-β1, TGF-β2, BMP-4, and BMP-7 in TAMs and the expression of vasohibin-1, vascular endothelial growth factor-A (VEGF-A), and VEGF-C in two pancreatic cancer cell lines (a nonmetastatic cell line Panc-1 and a distant metastatic cell line HPAF-II) were measured by real-time reverse transcription-polymerase chain reaction (RT-PCR). The TGF-β receptor 1 and BMP receptor 1 were inhibited by the inhibitor SB-431542 and LDN193189, respectively. Thereafter, vasohibin-1, VEGF-A, and VEGF-C expression was detected by real-time RT-PCR. We found that the expression of TGF-β1, TGF-β2, BMP-4, and BMP-7 was upregulated in TAMs cocultured with pancreatic cancer cells. Vasohibin-1, VEGF-A, and VEGF-C mRNA expression in pancreatic cancer cells was upregulated by TAMs. Vasohibin-1 expression in pancreatic cancer cells cocultured with TAMs was upregulated significantly when TGF-β receptors or BMP receptors were inhibited, but VEGF-C expression was downregulated. Therefore, Vasohibin-1 expression is regulated by the TGF-β/BMP signaling between TAMs and pancreatic cancer cells. These results might shed a new light on the antiangiogenesis therapy in the pancreatic cancer.

Monitoring Current-Season Spread of Potato virus Y in Potato Fields Using ELISA and Real-Time RT-PCR.

The current-season spread of Potato virus Y (PVY) was investigated in New Brunswick, Canada, in 11 potato fields planted with six different cultivars in 2009 and 2010. In all, 100 plants selected from each field were monitored for current-season PVY infections using enzyme-linked immunosorbent assay (ELISA) and real-time reverse-transcription polymerase chain reaction (RT-PCR) assay. Average PVY incidence in fields increased from 0.6% in 2009 and 2% in 2010 in the leaves to 20.3% in 2009 and 21.9% in 2010 in the tubers at the time of harvest. In individual fields, PVY incidence in tubers reached as high as 37% in 2009 and 39% in 2010 at the time of harvest. Real-time RT-PCR assay detected more samples with PVY from leaves than did ELISA. A higher number of positive samples was also detected with real-time RT-PCR from growing tubers compared with the leaves collected from the same plant at the same sampling time. PVY incidence determined from the growing tubers showed a significant positive correlation with the PVY incidence of tubers after harvest. Preharvest testing provides another option to growers to either top-kill the crop immediately to secure the seed market when the PVY incidence is low or leave the tubers to develop further for table or processing purposes when incidence of PVY is high.

Matrix Metalloproteinase 13 Expression in Response to Double‐Stranded RNA in Human Chondrocytes

To investigate the mechanism of matrix metalloproteinase 13 (MMP-13) expression in chondrocytes via pattern-recognition receptors (PRRs) for double-stranded RNA (dsRNA). Differential expression of PRRs was determined by real-time reverse transcription-polymerase chain reaction (RT-PCR) of RNA from patients with osteoarthritis (OA) and patients with femoral neck fracture (as normal control). Isolated human articular chondrocytes and the chondrosarcoma cell line SW-1353 were activated with poly(I-C) of different molecular weights as a dsRNA mimic, and changes in gene and protein expression were monitored by real-time RT-PCR and immunoblotting, respectively. The dsRNA signaling moieties Toll-like receptor 3 (TLR-3), retinoic acid-inducible gene 1 (RIG-1), and nucleotide-binding oligomerization domain-like receptor X1 were all differentially expressed in OA cartilage compared to normal cartilage, as determined by gene expression screening. Depletion of the dsRNA-sensing receptors TLR-3, RIG-1, or melanoma differentiation-associated gene 5 (MDA-5) suppressed the induction of MMP13 messenger RNA (mRNA) expression by poly(I-C), regardless of its mode of delivery. In addition, depletion of the downstream transcription factor interferon regulatory factor 3 resulted in reduced induction of MMP13 mRNA expression by poly(I-C). Signaling by dsRNA in chondrocytes requires a range of PRRs, including TLR-3, RIG-1, and MDA-5, for the full-induction of MMP13, thus providing tight regulation of a gene critical for maintenance of cartilage integrity. Our data add to the understanding of MMP13 regulation, which is essential before such mechanisms can be exploited to alleviate the cartilage destruction associated with OA.

Circulation of bluetongue virus in goats in the Karamoja region of Uganda

The presence of bluetongue virus (BTV) in indigenous goats from the Karamoja region of northern Uganda was investigated. A total of 300 goats were sampled (serum and whole blood) from five districts within the Karamoja region. The samples were analysed for the presence of bluetongue (BT) antibodies using a commercial Enzyme-linked immunosorbent assay (ELISA) and for the presence of BTV viral RNA by real-time Reverse transcription polymerase chain reaction (RT-PCR), because BTV is an RNA virus. Of the 300 goats tested, 269 (90%) were positive for BTV antibodies, indicating high levels of BTV circulation within the region. Out of the 150 whole blood samples tested for the presence of the virus by real-time RT-PCR, 84 (56%) were positive for BTV RNA. This study, which is the first of its kind in Uganda, showed a high seroprevalence of BT antibodies and active circulation of BTV in a high proportion of goats in the Karamoja region.

CD24 and CD44 in salivary gland pleomorphic adenoma and in human salivary gland morphogenesis: differential markers of glandular structure or stem cell indicators?

Salivary gland neoplasms originate from salivary gland compartments, to which they are histologically related. Pleomorphic adenoma (PA) is a benign salivary gland neoplasm that comprises epithelial and myoepithelial cells and a complex stroma, whose structure, architecture and origin (from intercalated ducts) suggest stem cell participation. We compared the expression of CD24 and CD44 in PA and in developing human salivary glands to investigate whether these markers can be considered as cancer stem cell markers. One hundred and one cases of PA and salivary gland specimens from 20 human fetuses were examined by immunohistochemistry and real-time reverse transcription polymerase chain reaction (RT-PCR). All PAs were positive for CD24 and CD44 by immunohistochemistry: neoplastic luminal structures were positive for CD24; modified myoepithelial cells were positive for CD44. In fetal salivary glands, these markers were restricted to the intercalated duct region. Real-time RT-PCR assays detected increased expression of CD44, but not CD24, in PA specimens in comparison with normal salivary gland controls. PA and stem cells share the expression of CD24 and CD44; their value as markers of neoplastic cell multipotency and the implications of their expression for tumour behaviour are yet to be determined.

Nuclear Accumulation of Prohibitin 1 in Osteoarthritic Chondrocytes Down‐Regulates PITX1 Expression

To decipher the molecular mechanisms down-regulating PITX1 expression in primary osteoarthritis (OA). The functional activity of different PITX1 promoter regions was assessed by luciferase reporter assay. Tandem mass spectrometry coupled to protein sequencing was performed using nuclear extracts prepared from OA chondrocytes, in order to identify proteins bound to DNA regulatory elements. Expression analyses of selected candidate proteins were performed by real-time reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry methods, using cartilage sections and articular chondrocytes from non-OA control subjects and patients with OA. Gain-of-function and loss-of-function experiments were performed in normal and OA chondrocytes, respectively, to study their effects on PITX1 regulation. The results were validated by real-time RT-PCR and immunohistochemistry in STR/Ort mice, a well-known animal model of OA. PITX1 promoter analyses led to the identification of prohibitin 1 (PHB1) bound to a distal E2F1 transcription factor site. Aberrant accumulation of PHB1 was detected in the nuclei of OA articular chondrocytes, and overexpression of PHB1 in control cells was sufficient to inhibit endogenous PITX1 expression at the messenger RNA and protein levels. Conversely, knockdown of PHB1 in OA articular chondrocytes resulted in up-regulation of PITX1. Studies of early molecular changes in STR/Ort mice revealed a similar nuclear accumulation of PHB1, which correlated with Pitx1 repression. Collectively, these data define an unrecognized role for PHB1 in repressing PITX1 expression in OA chondrocytes.

Polycistronic transcription of fused cassettes and identification of translation initiation signals in an unusual gene cassette array from Pseudomonas aeruginosa.

The gene cassettes found in class 1 integrons are generally promoterless units composed by an open reading frame (ORF), a short 5' untranslated region (UTR) and a 3' recombination site ( attC). Fused gene cassettes are generated by partial or total loss of the attC from the first cassette in an array, creating a fusion with the ORF from the next cassette. These structures are rare and little is known about their mechanisms of mobilization and expression. The aim of this study was to evaluate the dynamic of mobilization and transcription of the gcu14- blaGES-1/ aacA4 gene cassette array, which harbours a fused gene cassette represented by blaGES-1/ aacA4. The cassette array was analyzed by Northern blot and real-time reverse transcription-polymerase chain reaction (RT-PCR) in order to assess the transcription mechanism of blaGES-1/ aacA4 fused cassette. Also, inverse polymerase chain reactions (PCR) were performed to detect the free circular forms of gcu14, blaGES-1 and aacA4. The Northern blot and real time RT-PCR revealed a polycistronic transcription, in which the fused cassette blaGES-1/ aacA4 is transcribed as a unique gene, while gcu14 (with a canonical attC recombination site) has a monocistronic transcription. The gcu14 cassette, closer to the weak configuration of cassette promoter (Pc), had a higher transcription level than blaGES-1/ aacA4, indicating that the cassette position impacts the transcript amounts. The presence of ORF-11 at attI1, immediately preceding gcu14, and of a Shine-Dalgarno sequence upstream blaGES-1/ aacA4 composes a scenario for the occurrence of array translation. Inverse PCR generated amplicons corresponding to gcu14, gcu14- aacA4 and gcu14- blaGES-1/ aacA4 free circular forms, but not to blaGES-1 and aacA4 alone, indicating that the GES-1 truncated attC is not substrate of integrase activity and that these genes are mobilized together as a unique cassette. This study was original in showing the transcription of fused cassettes and in correlating cassette position with transcription.

Evaluation of virus isolation, one-step real-time reverse transcription polymerase chain reaction assay, and two rapid influenza diagnostic tests for detecting canine Influenza A virus H3N8 shedding in dogs

Sustained transmission of canine Influenza A virus (CIV) H3N8 among U.S. dogs underscores the threat influenza continues to pose to canine health. Because rapid and accurate detection of infection is critical to the diagnosis and control of CIV, the 2 main objectives of the current study were to estimate and compare the sensitivities of CIV testing methods on canine swab samples and to evaluate the performance of Flu Detect™ (Synbiotics Corp., Kansas City, MO) for detecting CIV nasal shedding in high-risk shelter dogs. To address the first objective, nasal and pharyngeal swab samples were collected from 124 shelter and household dogs seen by Colorado State University Veterinary Teaching Hospital clinicians for canine infectious respiratory disease between April 2006 and March 2007 and tested for CIV shedding using virus isolation, the rapid influenza diagnostic test Directigen Flu A+B™ (BD Diagnostic Systems, Sparks, MD), and real-time reverse transcription polymerase chain reaction (RT-PCR). For the second objective, 1,372 dogs with unknown respiratory health status were sampled from 6 U.S. shelters from December 2009 to November 2010. Samples were tested for presence of CIV using real-time RT-PCR and Flu Detect. Using a stochastic latent class modeling approach, the median sensitivities of virus isolation, rapid influenza diagnostic test, and real-time RT-PCR were 72%, 65%, and 95%, respectively. The Flu Detect test performed poorly for detecting CIV nasal shedding compared to real-time RT-PCR. In conclusion, the real-time RT-PCR has the highest sensitivity for detecting virus nasal shedding and can be used as a rapid diagnostic test for CIV.

A one-step multiplex real-time RT-PCR assay for rapid and simultaneous detection of human norovirus genogroup I, II and IV

A one-step multiplex real-time reverse transcription-PCR (RT-PCR) assay was developed for one-tube and simultaneous detection of three genogroups of human norovirus, genogroup I, II and IV (GI, GII and GIV). The specificity and sensitivity of the assay were evaluated and 50 samples were tested by using this assay. The results showed that the multiplex assay had high sensitivity and specificity. The amplification efficiencies of the assay were 91.3%, 90.1%, 88.9% and the detection limits were up to 16.9, 6.3, 43.0 copies/reaction respectively for norovirus GI, GII and GIV detection. No cross-reaction with the other examined RNA viruses was observed, and the qualitative analysis of samples showed that the multiplex assay had a good consistency with its corresponding monoplex assays for the detection of norovirus GI, GII and GIV (Kappa values were 0.848, 0.876 and 0.812 respectively).