Duplex RT-PCR Research Articles

Detection of Mixed Infection of Tobamoviruses in Tomato and Bell Pepper by using RT-PCR and Duplex RT-PCR

Detection of Mixed Infection of Tobamoviruses in Tomato and Bell Pepper by using RT-PCR and Duplex RT-PCR

Evaluation of Feline Coronavirus Viraemia in Clinically Healthy and Ill Cats with Feline Infectious Peritonitis

Feline Coronavirus (FCoV) comprises virulent and avirulent biotypes. While both biotypes can enter the bloodstream of a cat, only the virulent biotypes would replicate in monocytes and macrophages and develop a fatal disease known as Feline Infectious Peritonitis (FIP). In the present study, FCoV viraemia was evaluated in 50 cats consisting of 40 overtly healthy and 10 ill cats suspected of FIP. The blood samples were screened for FCoV genomic RNA by a RT-PCR assay and then followed by a duplex RT-PCR for detection of replicating viral mRNA. In the healthy cats, the virus and its replicating mRNA were detected in 67.5 and 15%, respectively. The later finding suggested that the virus was replicating in a few cats with no clinical sign shown and indicated that FCoV viraemia do not necessarily lead to FIP. Probably the avirulent virus does multiply at low level in the blood or cat can harbor the virulent virus in an early stage of FIP without clinical signs yet. In FIP-suspected cases, all of the ill cats were positive for both FCoV and the replicating viral mRNA suggesting that FCoV could have replicated in blood and produced high amount of the virus and its components which were detectable by the both assays. The duplex RT-PCR assay which has been used to detect the replicating viral mRNA in blood was more specific than the general screening RT-PCR test for the diagnosis of FIP. The RT-PCR results however, should be interpreted in conjunction with other clinical symptoms.

Rapid and Sensitive Detection of Bovine Coronavirus and Group A Bovine Rotavirus from Fecal Samples by Using One-Step Duplex RT-PCR Assay

Bovine coronavirus (BCoV) and group A bovine rotavirus (BRV) are two of major causes for neonatal calf diarrhea. In the present study, a one-step duplex RT-PCR was established to detect and differentiate BCoV and group A BRV from fecal samples. The sensitivity of this method for BCoV and group A BRV was 10 PFU/100 µl and 1 PFU/100 µl, respectively. Twenty-eight diarrhea fecal samples were detected with this method, the result showed that 2 samples were identified as co-infected with BCoV and group A BRV, 26 samples were group A BRV positive, and 2 samples were negative. It proved that this method is sensitive for clinical fecal samples and is worth applying to laboratory diagnosis for BCoV and group A BRV.

Alterações clínicas e patológicas em ovinos infectados naturalmente pelo vírus da língua azul no Rio Grande do Sul

Língua azul (LA) é uma doença causada pelo vírus da língua azul (VLA) e transmitida por vetores do gênero Culicoides. Estudos sorológicos têm demonstrado a ampla presença do vírus no Brasil; entretanto, informações clínicas da LA na América do Sul são limitadas. Esse trabalho descreve alterações clínico-patológicas em ovinos acometidos pela LA no Sul do Brasil. Em dois surtos, em propriedades distintas, 15 ovinos apresentaram como principais sinais clínicos hipertermia, apatia, aumento de volume da face e região submandibular, dificuldade de deglutição com regurgitação, secreção nasal mucopurulenta esverdeada, alterações respiratórias, além de acentuada perda de peso e erosões na mucosa oral. Os achados de necropsia em seis ovinos afetados incluíram edema subcutâneo na face e região ventral do tórax, secreção nasal esverdeada, esôfago dilatado preenchido por grande quantidade de conteúdo alimentar, pulmões não colabados com áreas consolidadas anteroventrais, bem como luz da traquéia e brônquios preenchida por espuma misturada com conteúdo alimentar. No coração e base da artéria pulmonar, havia focos de hemorragia. Histologicamente, as principais alterações observadas ocorriam no tecido muscular cardíaco e esquelético, especialmente no esôfago e consistiam de lesões bifásicas caracterizadas por degeneração/necrose hialina e flocular de miofibras associadas com micro-calcificação e infiltrado inflamatório mononuclear. Pneumonia aspirativa associada à presença de material vegetal e bactérias na luz de brônquios também foi observada. O diagnóstico de LA foi confirmado pela detecção do genoma viral por duplex RT-PCR em amostras de sangue de animais afetados, seguido da identificação do VLA, sorotipo 12 por sequenciamento.

Validation of commercial real-time RT-PCR kits for detection of influenza A viruses in porcine samples and differentiation of pandemic (H1N1) 2009 virus in pigs

Swine influenza, apart from its importance in animal health, may also be of public health significance. Although the first human infections with the multi-reassortant H1N1 virus (pH1N1/09) responsible for the 2009 pandemic were not related to pig exposure, this virus was shown to be related genetically to swine influenza viruses (SIV) and easily transmissible to pigs. In addition to direct animal health concerns, transmission and possible adaptation of the pH1N1/09 virus in pigs may have serious consequences on the risk of human infection by increasing the reservoir of this virus and the risk of possible emergence of new reassortant viruses with increased virulence for pigs and/or humans. Sensitive tools to monitor and detect rapidly such an infection are therefore mandatory. In this study, five commercial real-time RT-PCR assays developed by manufacturers LSI and Adiagène were assessed and validated, (i) for rapid detection of influenza A viruses, including pH1N1/09, in pig and (ii) for the differentiation of pH1N1/09 in that species. Two kits target the influenza A virus M gene, two others amplify the pH1N1/09 virus H1 gene and one kit targets the pH1N1/09 virus N1 gene. All five kits are ready-to-use, one-step duplex RT-PCR and contain an internal positive control (IPC), appropriate for porcine biological samples, for assessing RNA extraction efficiency and the presence of PCR inhibitors. They have been used successfully by veterinary laboratories and shown to be powerful tools for the diagnosis and epidemiological surveillance of influenza virus infections in pigs.

Apple juice intervention modulates expression of ARE-dependent genes in rat colon and liver

The risk of cancer and other degenerative diseases is inversely correlated with consumption of fruits and vegetables. This beneficial effect is mainly attributed to secondary plant constituents such as polyphenols, supposed to play a major role in protection against ROS (reactive oxygen species)-associated toxicity. To elucidate the potential of differently manufactured apple juices (clear AJ/cloudy AJ/smoothie, in comparison with a polyphenol-free control juice) to modulate expression of ARE-dependent genes. In male Sprague-Dawley rats (n=8/group; 10d juice intervention, 4d wash-out; 4 treatment cycles), expression of target genes (superoxide dismutase, SOD1/SOD2; glutathione peroxidase, GPX1/GPX2; γ-glutamylcysteine ligase, GCLC/GCLM; glutathione reductase, GSR; catalase, CAT; NAD(P)H:quinone oxidoreductase-1, NQO1 and transcription factor erythroid-derived 2-like-2, Nrf2) was quantified with duplex RT-PCR, using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as control. In colon and liver of rats consuming polyphenol-free control juice, rather similar basic expressions were observed (relative GAPDH ratios ranging from 2 to 0.7 and 2.5-0.3, respectively). In the distal colon, apple juice intervention slightly but significantly induced most genes (e.g. GPX2, GSR, CAT, Nrf2; p<0.001), whereas in the liver only GPX1 and NQO1 mRNA were up-regulated; other hepatic target genes were not affected or down-regulated (SOD1, SOD2, GCLC/M, GSR), concomitant with the absence of Nrf2 induction. Induction of antioxidant gene expression differed with juice type (cloudy AJ>clear AJ~smoothie). Taken together, the results underline the potential of polyphenol-rich apple juice to increase the expression of ARE-dependent antioxidant genes.

Simultaneous Detection of Mixed Infection of Onion yellow dwarf virus and an Allexivirus in RT-PCR for Ensuring Virus Free Onion Bulbs.

Reduced seed production in onion is associated with Onion yellow dwarf virus (OYDV), a filamentous Potyvirus. Onion is also infected with other filamentous virus particles suspected to be Allexivirus. RT-PCR was used to detect mixed infection of both the viruses in leaves and bulbs. A duplex RT-PCR was developed, which simultaneously detected the presence of these two viruses in winter (Rabi) onion bulb. In summer (Kharif) onion bulbs only Allexivirus was detected. The absence of OYDV in summer crop is discussed. The sequencing of RT-PCR amplified products confirmed the identity of OYDV and Allexivirus, the latter showing closer identity to Garlic virus C (GVC)/Garlic mite-borne mosaic virus. This makes the first detection of an Allexivirus in onion crop in India. The duplex RT-PCR to detect these viruses (OYDV and Allexivirus) would be an improvement for indexing of viruses in onion bulbs for seed production.

The identification of the first isolate of influenza B virus using a duplex RT-PCR DNA sequencing in Saudi Arabia (B/Riyadh/01/2007)

Although Influenza virus infections threaten thousands of lives each year worldwide and bear the risk of been an epidemic, little is known about the circulating strains in Saudi Arabia. The availability of a rapid and accurate diagnostic approach is essential for effective treatment and disease control. In the current study, we optimized a duplex RT-PCR assay for the simultaneous detection of influenza A and B viruses in clinical samples. The developed assay was utilized for testing 100 nasopharyngeal aspirates collected from young children hospitalized with acute respiratory tract infection in Riyadh. Influenza B not A viruses, were identified as possible causes of the disease syndrome. Recovery of influenza B virus from clinical samples was achieved by serial passage in chicken embryos and the virus isolate was designated as Influenza B/Riyadh/01/2007. The identity of B/Riyadh/01/2007 was confirmed by sequencing the RT-PCR product. The sequenced data was submitted to the Gene Bank under the accession number GU135839. An almost complete homology was recognized with all Influenza B virus strains available on Gene Bank. Although several studies suggested the presence of influenza B virus in respiratory tract infection of Saudi children, this is the first report that describes the isolation and partial identification of the circulating virus. Further studies that fully characterize the isolated strain are ongoing.

Prospective efficacy of molecular preoperative diagnostics of papillary thyroid carcinoma

Background. The diagnostic efficacy of the molecular analysis that included determination of papillary thyroid carcinoma (PTC) marker gene expression levels and BRAF mutation in fine-needle aspiration biopsy material was evaluated in a prospective study of patients with thyroid nodules. Materials and methods. Totally, 36 patients (29 females and 7 males) with thyroid nodules were included in the study. The mRNA expression of genes (SFTPB and TFF3) was estimated in relation to a housekeeping gene level (KPNA4) by means of duplex RT-PCR followed by the band intensity measurement. Detection of BRAF mutation was performed by PCR followed by direct sequencing. Results. In 25/32 (78.1%) cases, results of the molecular test were in agreement with the cytological diagnosis (7/7 PTC and 18/25 non-PTC) further confirmed by histological examination of tissues surgically removed from all seven PTC patients and 10 individuals with benign nodules. In 7/32 patients (21.9%) there was a discrepancy between cytological findings and molecular results, which revealed a benign nodule and a PTC-like pattern, respectively. Upon a repeated examination of five of these patients about one year later, three were cytologically diagnosed with PTC (all patients had been operated on), and the diagnosis of the other two patients remained unchanged. Conclusions. The results have demonstrated that the molecular analysis of FNAB material is an informative means of the preoperative diagnosis of thyroid nodules as it allows identification of patients with suspected PTC before other diagnostically significant changes take place. Keywords: papillary thyroid carcinoma, fine-needle aspiration biopsy, preoperative diagnosis, molecular test

Toxoplasma gondii: Simple duplex RT-PCR assay for detecting SAG1 and BAG1 genes during stage conversion in immunosuppressed mice

Toxoplasmic encephalitis (TE) is caused by reactivation of dormant bradyzoites into rapidly dividing tachyzoites of the apicomplexan parasite Toxoplasma gondii in immune-compromised hosts. Diagnosis of this life-threatening disease is complicated, since it is difficult to distinguish between these two stages. It is, therefore, mainly based on a test positive for T. gondii antibodies, and specific clinical symptoms. We developed a duplex RT-PCR to detect the expression of bradyzoite (BAG1) and tachyzoite (SAG1) specific genes simultaneously during tachyzoite/bradyzoite stage conversion. The conversion reaction was observed in many organs of experimental mice, indicated by tachyzoites in the cerebrum, cerebellum, heart and lung, beginning in week 1 after the suppression period, and continuing until the end. Bradyzoites were also detected in nearly all organs throughout the study, suggesting that during the reactivation period, bradyzoites not only escape from cysts and reinvade neighboring cells as tachyzoites, but are also driven into developing new bradyzoites. The results of our study show that duplex RT-PCR is an easy, rapid, sensitive, and reproducible method, which is particularly valuable when numerous samples must be analyzed. This technique may usefully serve as an alternate tool for diagnosing TE in severely immunocompromised patients.

Differential expression of VEGFA, TIE2, and ANG2 but not ADAMTS1 in rat mesenteric microvascular arteries and veins

Microvessels respond to metabolic stimuli (e.g. pO(2)) and hemodynamic forces (e.g. shear stress and wall stress) with structural adaptations including angiogenesis, remodeling and pruning. These responses could be mediated by differential gene expression in endothelial and smooth muscle cells. Therefore, rat mesenteric arteries and veins were excised by microsurgery, and mRNA expression of four angioadaptation-related genes was quantified by real time duplex RT-PCR in equal amounts of total RNA, correlated to two different house keeping genes (beta-actin, GAPDH). The results show higher expression of VEGFA, TIE2, and ANG2 in arteries than in veins, but equal expression of ADAMTS1. Higher availability of VEGFA mRNA in endothelial cells of arteries shown here could contribute to the maintenance of mechanically stressed blood vessels and counteract pressure-induced vasoconstriction.

Simultaneous detection of Japanese yam mosaic virus and Yam mild mosaic virus from yam leaves using a tube capture reverse transcription-polymerase chain reaction assay

To detect Japanese yam mosaic virus (JYMV) and Yam mild mosaic virus (YMMV) in yam plants in Japan, we developed a duplex RT-PCR assay consisting of a tube-capture procedure followed by one-step RT-PCR with two primer pairs. A 241-bp fragment of the coat protein region of JYMV and a 174-bp fragment of the nuclear inclusion protein b region of YMMV were amplified, thus identifying the two viruses from yam plants cultivated in Yamaguchi Prefecture in 2007. All water yam plants examined were infected with YMMV alone. All the Japanese yam and Chinese yam plants were infected with either JYMV alone or both JYMV and YMMV, suggesting that YMMV and JYMV are prevalent among field-grown yam plants.

Mise au point d’une technique de RT-PCR duplex en temps réel pour la détection des virus influenza A et B

Influenza viruses continue to be accountable for winter outbreaks. The potential for developing complications is higher in certain risk groups, such as children, the elderly and individuals with chronic medical conditions. We have presented a duplex real-time RT-PCR for detection of influenza A and B. The sensibility of our method was estimated at 0.01TCID(50)/ml for detection of influenza A and 0.1TCID(50)/ml for influenza B. Any other viruses with respiratory tract tropism were detected with our method. After that, we tested our duplex RT-PCR on 119 samples (nasal aspirates and liquids of broncho-alveolar washes) collected in the CHRU of Lille between November 2005 and January 2006 from patients aged one month to 81 years. Conventional methods such as direct immunofluorescence (IF) assay and/or cell culture were applied on these samples. Four samples were positive for influenza A virus detection and, in particular, one liquid of broncho-alveolar wash for which the direct IF assay was negative. Thus, our method is adapted to diagnosis of flu infections. Nevertheless, our duplex RT-PCR will be tested during a flu outbreak.

Simultaneous detection of Onion yellow dwarf virus and Shallot latent virus in infected leaves and cloves of garlic by duplex RT-PCR.

A duplex RT-PCR was standardized for the simultaneous detection of Onion yellow dwarf virus (OYDV) and Garlic latent virus (GarLV), a synonym for the recognized species Shallot latent virus (SLV) in garlic, which allowed the successful identification of both viruses in cloves and leaves. The titre of OYDV was higher both in leaves and bulbs compared with SLV. In the leaves, OYDV was detected up to an RNA dilution of 10-4 while SLV could be detected up to an RNA dilution of 10-3. Duplex RT-PCR detected both viruses in ten commercial garlic cultivars. The procedure is costeffective and sensitive and will be highly useful in quarantine and certification programmes.

Inter-laboratory evaluation of a duplex RT-PCR method using crude extracts for the simultaneous detection of Prune dwarf virus and Prunus necrotic ringspot virus

The operational capacity of a duplex RT-PCR method for simultaneous detection of Prune dwarf virus (PDV) and Prunus necrotic ringspot virus (PNRSV) has been established by nine European laboratories. A total of 576 samples from Prunus trees with known sanitary status, corresponding to 32 samples in two repetitions for each laboratory, were analysed. The level of sensitivity achieved by the method was 98.3% for PDV and 90.4% for PNRSV. The specificity was 87.4% for PDV and 94.3% for PNRSV. The unilateral 95% confidence intervals were calculated for all these values. Cohen’s Kappa coefficient of repeatability and reproducibility of the technique indicated a strong agreement between data. Likelihood ratios were 7.50 (positive) and 0.02 (negative) for PDV. For PNRSV, the positive likelihood ratio was 15.00 while the negative likelihood ratio was 0.11. In addition, post-test probabilities of infection were calculated to manage the risk associated with the routine use of this method. This allows an accurate test result interpretation to facilitate the integration of this new technique into a certification scheme.

R031: The Expression of Niban Is Up-Regulated in HNSCC

Niban is well expressed in Eker rat renal carcinogenesis and as well as human renal carcinoma. The expression of Niban was up-regulated in early pre-neoplastic lesions in the renal carcinogenesis models. Thus Niban is a candidate as a marker for renal carcinoma. In addition other human carcinomas were stained for Niban. However the role of Niban is still unknown. Head and neck squmamous cell carcinoma (HNSCC) represents the sixth most common cancer worldwide. We examined Niban expression in HNSCC by immunohistochemical staining using polyclonal rabbit antihuman Niban antibody. Normal head and neck epithelium was not stained for Niban, but all 43 HNSCC cases were positively stained for Niban and dysplasia in 14/22 (63%) were weakly stained for Niban. RT-PCR verified the Niban expression in HNSCC at the messenger RNA level. Niban may not only play an important role in the human head and neck carcinogenesis but also is a candidate as a good molecular marker of the HNSCC. Reverse transcriptase-polymerase chain reaction Duplex RT-PCR analysis for Niban together with the internal control GAPDH gene showed bands of Niban and GAPDH in heart, normal lymph nodes, and 3 cases of HNSCC. These 3 cases were immunostained for Niban. Three cases of normal head and neck tissue only showed signals for GAPDH, but not for Niban. The results verified the Niban expression in HNSCC at the messenger RNA level. (Table. 3)

Inhibition of mosaic disease of Gladiolus caused by Bean yellow mosaic- and Cucumber mosaic viruses by virazole

A method for the establishment of callus tissue from infected Gladiolus ( Gladiolus psittacinus var. Hookeri cv. Red) corms, regeneration and elimination of viruses, viz. Bean yellow mosaic virus (BYMV) and Cucumber mosaic virus (CMV) from callus tissue was developed. The callus tissue, from infected central bud slices of cormels was raised and subjected to various concentrations of virazole (ribavirin, 1-β- d-ribofuranosyl-1, 2, 4-triazole-3-carboxamide) for elimination of viruses. The tissues were screened for the presence of virus (es) after culturing them on to the virazole supplemented media for a period of 6–8 weeks which successfully eliminated both BYMV and CMV in callus tissue cultures of Gladiolus. The tissues were then differentiated into plantlets/corms and tested for the presence/absence of virus (es) employing serological (electroblot immunoassay, EBIA) and nucleic acid-based (nucleic acid spot hybridization, NASH and reverse transcriptase polymerase chain reaction, RT-PCR) techniques. Additionally, duplex RT-PCR was also developed for simultaneous detection of BYMV and CMV in Gladiolus tissues at various developmental stages. The tissues were regenerated and certified virus-free corms/Gladiolus plantlets were produced.

A duplex RT-PCR assay for detection of genome segment 7 (VP7 gene) from 24 BTV serotypes

Since 1998, six distinct serotypes of Bluetongue virus (BTV) have invaded Southern and Central Europe, persisting in some regions for up to 6 years and resulting in the deaths of >1.8 million sheep. Rapid and reliable methods of virus detection and identification play an essential part in our fight against bluetongue disease (BT). We have therefore developed and evaluated a duplex, one-step RT-PCR assay that detects genome segment 7 (encoding the major serogroup (virus-species) specific antigen and outer-core-protein VP7) from any of the 24 BTV serotypes. Although Seg-7 is highly conserved, there are sequence differences in the near terminal regions that identify two distinct phylogenetic groups. Two sets of primers (targeting Seg-7 terminal regions of viruses from these two groups) were included in a duplex RT-PCR assay system. Assay sensitivity was evaluated using tissue culture derived virus, infected vector insects and clinical samples (blood and other tissues). The assay reliably amplified Seg-7 from any of the BTV strains tested, including isolates of the 24 BTV serotypes and isolates from different geographic origins. No cross-reactions were detected with members of closely related Orbivirus species ( African horsesickness virus (AHSV), Epizootic haemorrhagic disease virus (EHDV), Equine encephalosis virus (EEV) and Palyam virus (PALV)).

Enhanced Reverse Transcription-PCR Assay for Detection of Norovirus Genogroup I

We have developed a one-tube reverse transcription (RT)-PCR method using the real-time TaqMan PCR system for the detection of norovirus genogroup I (NV GGI). By introduction of a novel probe based on locked nucleic acid technology, we enhanced the sensitivity of the assay compared to those of conventional TaqMan probes. The sensitivity of the NV GGI RT-PCR was determined by probit analysis with defined RNA standards and quantified norovirus isolates to 711 copies/ml (95% detection limit). In order to detect PCR inhibition, we included a heterologous internal control (IC) system based on phage MS2. This internally controlled RT-PCR was tested on different real-time PCR platforms, LightCycler, Rotorgene, Mastercycler EP realplex, and ABI Prism. Compared to the assay without an IC, the duplex RT-PCR exhibited no reduction in sensitivity in clinical samples. In combination with an established NV GGII real-time RT-PCR, we used the novel assay in a routine assay for diagnosis of clinical and food-borne norovirus infection. We applied this novel assay to analyze outbreaks of nonbacterial acute gastroenteritis. Norovirus of GGI was detected in these outbreaks. Sequence and similarity plot analysis of open reading frame 1 (ORF1) and ORF2 showed two genotypes, GGI/2 and GGI/4, in semiclosed communities.

Rapid, reliable and inexpensive quality assessment of biotinylated cRNA

The interpretation of oligonucleotide array experiments depends on the quality of the target cRNA used. cRNA target quality is assessed by quantitative analysis of the representation of 5' and 3' sequences of control genes using commercially available Test arrays. The Test array provides an economically priced means of determining the quality of labeled target prior to analysis on whole genome expression arrays. This manuscript validates the use of a duplex RT-PCR assay as a faster (6 h) and less expensive (<10 US dollars) but equally accurate alternative to the Test arrays in determining biotinylated cRNA quality. Forty-one different cRNA samples were hybridized to HG-U133A microarrays from Affymetrix. Ten cRNA samples with a beta-actin 3'/5' ratio >6 were chosen and classified as degraded cRNAs, and 31 samples with a beta-actin 3'/5' ratio <6 were selected as good quality cRNAs. Blinded samples were then used for the RT-PCR assay. After gel electrophoresis, optical densities of the amplified 3' and 5' fragments of beta-actin were measured and the 3'/5' ratio was calculated. There was a strong correlation (r(2) = 0.6802) between the array and the RT-PCR beta-actin 3'/5' ratios. Moreover, the RT-PCR 3'/5' ratio was significantly different (P < 0.0001) between undegraded (mean +/- SD, 0.34 +/- 0.09) and degraded (1.71 +/- 0.83) samples. None of the other parameters analyzed, such as i) the starting amount of RNA, ii) RNA quality assessed using the Bioanalyzer Chip technology, or iii) the concentration and OD260/OD280 ratio of the purified biotinylated cRNA, correlated with cRNA quality.