Duplex Reverse Transcription-polymerase Chain Reaction Research Articles

Differentiation of virulent Shimen and vaccine C strains of classical swine fever virus by duplex reverse-transcription polymerase chain reaction

ABSTRACTIt is difficult to differentiate pigs that have been injected attenuated vaccines from those that are infected by a virulent virus. In this study, a duplex reverse transcription polymerase chain reaction (RT-PCR) was developed for simultaneous detection and identification of the pathogenic strain (Shimen-strain) and attenuated vaccine strain (C-strain) of classical swine fever virus (CSFV). A fragment of 438 bp or 265 bp was amplified from the genomic RNA of the Shimen-strain or the C-strain, respectively. Both types of viruses were simultaneously identified from the mixed samples of Shimen-strain and C-strain, while no PCR bands were obtained from the templates of some other porcine original viruses with the designed primers. The lowest limit of detection was 0.0616 pg for the C-strain RNA and 0.0844 pg for the Shimen-strain RNA. The developed RT-PCR was adopted to test CSFV from 65 collected samples. Compared with quantitative real-time PCR, the results indicated that the developed RT-PCR can not only be used to distinguish the virulent and attenuated CSFV, but also possesses high sensitivity for detecting CSFV. This is a convenient method to detect and differentiate pigs infected with virulent CSFV from the pigs immunized with the C-strain.

Development and application of a universal and simplified multiplex RT-PCR assay to detect five potato viruses

Potato virus X (PVX), Potato virus Y (PVY), Potato leafroll virus (PLRV), Potato virus M (PVM), and Potato virus A (PVA) commonly cause mixed infections and major economic losses in potato crops, so a multiplex reverse transcription polymerase chain reaction (RT-PCR) assay has been greatly needed to detect all five viruses simultaneously. Previously published primers were evaluated, and five pairs of highly specific primers were selected for use. The quintuplex RT-PCR assay was optimized for primer concentration, annealing temperature, number of PCR cycles, and extension time. Finally, the quintuplex RT-PCR assay was successfully established. The amplified target fragments of 711 base pairs (bp) for PVX, 447 bp for PVY, 336 bp for PLRV, 520 bp for PVM and 273 bp for PVA, and the viral origins of the PCR products were confirmed by sequencing. This system can be easily customized for simplex, duplex, triplex, tetraplex, and quintuplex RT-PCRs by changing only the primer pairs while keeping the final reaction volume and the concentrations of other reagents constant to detect any combination of PVX, PVY, PLRV, PVM, and PVA viruses. In this way, the detection systems can be customized to the characteristics of viral infections specific to China’s potato-producing regions for excellent detection efficiency and meeting the needs of potato producers. The detection limit of this assay was up to 10−3 dilution in tetraplex and quintuplex RT-PCR, and even higher in simplex, duplex, and triplex RT-PCR. The efficiency of the quintuplex RT-PCR was compared with that of a DAS-enzyme-linked immunosorbent assay to detect the five viruses in 320 field potato samples, 195 in vitro plantlets and pooled potato samples. This new multiplex RT-PCR method is not only effective but also faster, cheaper, more sensitive, and more reliable than DAS-ELISA. It is especially suitable for the testing of in vitro plantlets and pooled samples of postharvest seed tubers.

A duplex RT-PCR assay for detection of H9 subtype avian influenza viruses and infectious bronchitis viruses

H9 subtype avian influenza virus (AIV) and infectious bronchitis virus (IBV) are major pathogens circulating in poultry and have resulted in great economic losses due to respiratory disease and reduced egg production. As similar symptoms are elicited by the two pathogens, it is difficult for their differential diagnosis. So far, no reverse transcription-polymerase chain reaction (RT-PCR) assay has been found to differentiate between H9 AIV and IBV in one reaction. Therefore, developing a sensitive and specific method is of importance to simultaneously detect and differentiate H9 AIV and IBV. In this study, a duplex RT-PCR (dRT-PCR) was established. Two primer sets target the hemagglutinin (HA) gene of H9 AIV and the nucleocapsid (N) gene of IBV, respectively. Specific PCR products were obtained from all tested H9 AIVs and IBVs belonging to the major clades circulating in China, but not from AIVs of other subtypes or other infectious avian viruses. The sensitivity of the dRT-PCR assay corresponding to H9 AIV, IBV and mixture of H9 AIV and IBV were at a concentration of 1×101, 1.5×101 and 1.5×101 50% egg infective doses (EID50) mL−1, respectively. The concordance rates between the dRT-PCR and virus isolation were 99.1 and 98.2%, respectively, for detection of samples from H9N2 AIV or IBV infected chickens, while the concordance rate was 99.1% for detection of samples from H9N2 AIV and IBV co-infected chickens. Thus, the dRT-PCR assay reported herein is specific and sensitive, and suitable for the differential diagnosis of clinical infections and surveillance of H9 AIVs and IBVs.

Detection and differentiation of wild-type and vaccine strains of canine distemper virus by a duplex reverse transcription polymerase chain reaction.

Canine distemper virus (CDV) is the cause of canine distemper (CD) which is a severe and highly contagious disease in dogs. In the present study, a duplex reverse transcription polymerase chain reaction (RT-PCR) method was developed for the detection and differentiation of wild-type and vaccine strains of CDV. Four primers were designed to detect and discriminate the two viruses by generating 638- and 781-bp cDNA products, respectively. Furthermore, the duplex RT-PCR method was used to detect 67 field samples suspected of CD from Guangdong province in China. Results showed that, 33 samples were to be wild-type-like. The duplex RT-PCR method exhibited high specificity and sensitivity which could be used to effectively detect and differentiate wild-type and vaccine CDV, indicating its use for clinical detection and epidemiological surveillance.

Microchip capillary electrophoresis with laser-induced fluorescence combined with one-step duplex reverse-transcription polymerase chain reaction for the rapid detection of Enterovirus 71 and Coxsackievirus A16 in throat swab specimens.

Enterovirus 71 and Coxsackievirus A16 are the main pathogens causing hand-foot-mouth disease. In this paper, microchip capillary electrophoresis with laser-induced fluorescence combined with one-step duplex reverse transcript-polymerase chain reaction has been developed for the detection of Enterovirus 71 and Coxsackievirus A16 in throat swab specimens. The specific reverse transcription-polymerase chain reaction amplicons labeled with SYBR Orange were separated by microchip capillary electrophoresis and detected by laser induced fluorescence detector within 7 min. The intraday and interday relative standard deviation of migration time for DNA Marker was in the range of 1.36-2.94 and 2.78-3.96%, respectively. The detection limits were as low as 2.06 × 10(3) copies/mL for Enterovirus 71 and 5 × 10(3) copies/mL for Coxsackievirus A16. No cross-reactivity was observed with rotavirus, astrovirus, norovirus, and adenovirus, which showed good specificity of the method. This assay was validated using 100 throat swab specimens that were detected by real-time reverse-transcript polymerase chain reaction in parallel and the two methods produced the same results. This study provided a rapid, sensitive and specific method for the detection of Enterovirus 71 and Coxsackievirus A16, which make a contribution to significant time and cost saving for the identification and treatment of patients.

Simultaneous detection of Apple Chlorotic Leaf Spot Virus and Apple mosaic virus in crab apples and apple rootstocks by duplex RT-PCR

Apple Chlorotic Leaf Spot Virus (ACLSV; family Betaflexiviridae, genus Trichovirus) and Apple mosaic virus (ApMV; family Bromoviridae, genus Ilarvirus) are economically important viruses of apple (Malus×domestica Borkh.). A duplex reverse transcription polymerase chain reaction (RT-PCR) assay was developed for simultaneous detection of both ACLSV and ApMV along with an internal control (NADH dehydrogenase subunit 5 gene). The internal control was used to minimize the risk of getting false negative results. Specific primers were designed against coat protein gene of ApMV while for ACLSV & internal control previously reported primer sets were used. Firstly the uniplex RT-PCR assay was standardized for the detection of each virus independently. Then duplex RT-PCR protocol for simultaneous detection of ACLSV and ApMV was standardized by using primer pairs ACLSV-CP1F & ACLSV-CP1R and ApMV-CP1F & ApMV-CP5R, respectively, along with primer set for internal control. Robustness of the technique was further validated wherein; duplex RT-PCR was carried out to detect both viruses in crab apples, rootstocks and different popular cultivars of apple. Results reveal that the duplex RT-PCR assay has detection sensitivity as that of uniplex RT-PCR assay for respective viruses. This optimized duplex RT-PCR provides a simple, rapid, sensitive and convenient way for simultaneous detection of ACLSV & ApMV by reducing the time and cost of the supplies.

A New Duplex Reverse Transcription PCR for Simultaneous Detection of Viable Cryptosporidium parvum Oocysts and Giardia duodenalis Cysts

To simultaneously detect viable Cryptosporidium parvum oocysts and Giardia duodenalis cysts for the purpose of reducing time and cost spent. A duplex reverse transcription polymerase chain reaction (RT-PCR) method was newly developed. Using duplex RT-PCR method for the hsp70 gene, viable (oo)cyst concentrations of 10(1) and 10(3) (oo)cysts/100 microL could be detected for C. parvum and G duodenalis, respectively. However, after heat-shock stimulation the expression of hsp70 mRNAs was detectable at 10(0) and 10(1) (oo)cysts/100 microL concentrations of C. parvum and G duodenalis, respectively. Thus, the detection sensitivity was significantly increased when the viable (oo)cysts were exposed to heat shock. This study describes a new duplex RT-PCR method for hsp70 gene to detect the viable (oo)cysts of the C. parvum and G duodenalis with less time consumed and at a lower cost. This newly developed duplex RT-PCR method may be used to detect these parasites not only in aquatic environments but also in clinical samples.

Duplex reverse transcription-polymerase chain reaction system to detect Potato spindle tuber viroid using an internal control mRNA and a non-infectious positive control RNA

An accurate and reliable system to detect Potato spindle tuber viroid (PSTVd) was developed based on duplex reverse transcription-polymerase chain reaction (RT-PCR) in combination with a plant mRNA internal control to evaluate the quality of RNA extracted from a dormant potato tuber and the effectiveness of the RT-PCR. In addition, a non-infectious RNA based on the PSTVd sequence was generated in vitro as a readily available positive control without the possibility of escape; this RNA was distinguishable from true positives in PSTVd infection. This detection system will be useful in plant quarantine and phytosanitary certification applications.

Comparison of the age-related porcine endogenous retrovirus (PERV) expression using duplex RT-PCR

Porcine endogenous retroviruses (PERVs) are members of family Retroviridae, genus Gamma retrovirus, and transmitted by both horizontally and vertically like other endogenous retroviruses (ERVs). PERV was initially described in the 1970s having inserted its gene in the host genome of different pig breeds, and three classes, PERV-A, PERV-B, and PERV-C are known. The therapeutic use of living cells, tissues, and organs from animals called xenotransplantation might relieve the limited supply of allografts in the treatment of organ dysfunction. Because of ethical considerations, compatible organ sizes, and physiology, the pig has been regarded as an alternative source for xenotransplantation. Sensitive duplex reverse transcription-polymerase chain reaction protocols for simultaneously detecting PERV gag mRNA and porcine glyceraldehydes 3-phosphate dehydrogenase mRNA in one tube was established. To compare the age-related PERV expression patterns of the lung, liver, spleen, kidney, heart, and pancreas in commercial pigs, 20 pigs from four age groups (5 heads each in 10 days-, 40 days-, 70 days-, and 110 days-old, respectively) were used in this study. The expression patterns of PERV were statistically different among age groups in lung, liver, and kidney (ANOVA, p < 0.05). These data may support in the selection of appropriate donor pigs expressing low levels of PERV mRNA.

Identification of four novel developmentally regulated gamma hemoglobin mRNA isoforms

In the course of developing assays for the molecular prediction of biological age, we serendipitously discovered four novel isoforms of gamma hemoglobin mRNA, designated HBG1n1, HBG1n2, HBG2n2, and HBG2n3, collectively termed HBGn isoforms. Here we report the molecular characterization and tissue expression of these isoforms. RNA obtained from human peripheral blood and various fetal and adult tissues was amplified with duplex reverse transcription polymerase chain reaction (RT-PCR) assays to determine the expression profiles of the HBGn isoforms. To determine if their molecular origin was either a genomic recombination or an undefined RNA rearrangement event, DNA and RNA samples were pretreated with RNaseI and/or DNaseI and then analyzed with the duplex RT-PCR assays. Alignment analysis indicated that the HBG1n(1/2) and HBG2n(2/3) isoforms were identical to the expected parental HBG1 or HBG2 sequences except for unique deletions that spanned the 3' end of exon two, the entire second intron, and the 5' end of exon three. RT-PCR duplex reactions revealed that the isoforms exhibit restricted expression to fetal hematopoietic tissue and newborn peripheral blood and appear to be temporally regulated during development. Finally, nucleic acid digestion experiments illustrate that the isoforms appear to be created by an RNA rearrangement event between penta- or octanucleotide direct repeats in adjacent exons. The precise genesis and function of these novel isoforms is unknown at present. Interestingly, each of the four isoforms identified maintain an open reading frame as well as 5' and 3' regulatory regions invoking the possibility that they encode a family of related polypeptides.

Development and evaluation of a 1-step duplex reverse transcription polymerase chain reaction for differential diagnosis of chikungunya and dengue infection

Dengue (DEN) and chikungunya (CHIK) have emerged as the 2 most important arboviral infections of global significance. The similarities in clinical presentations, their circulation in the same geographic area, and the transmission through the same vector necessitate an urgent need for the differential diagnosis of these 2 infections. So far, no single assay is reported for differential diagnosis of these 2 infections. In this study, we report the development and evaluation of a 1-step single-tube duplex reverse transcription polymerase chain reaction (D-RT-PCR) assay by targeting E1 gene of CHIK and C-prM gene junction of DEN virus (DENV), respectively. The sensitivity of this assay was found to be better than conventional virus isolation and could detect as low as 100 copies of genomic RNA, which is equivalent to respective virus-specific RT-PCR. The evaluation was carried out with 360 clinical samples from recent CHIK and DEN outbreaks in India. This assay could also be able to detect dual infection of CHIK and DEN in 3 patients. The phylogenetic analysis based on the nucleotide sequencing of D-RT-PCR amplicon could precisely identify the genotypes of all the serotypes of DENV and CHIK viruses (CHIKV). These findings demonstrate the potential clinical and epidemiologic application of D-RT-PCR for rapid sensitive detection, differentiation, and genotyping of DENV and CHIKV in clinical samples.

Duplex — reverse transcription — polymerase chain reaction (D-RT-PCR)-a technique for the simultaneous detection of viruses causing sugarcane mosaic

Sugarcane mosaic virus (SCMV) and Sugarcane streak mosaic virus (SCSMV) are the two major RNA viruses that are widely prevalent in most of the Asian countries including India and are the causative viruses of mosaic in sugarcane. We have established that these two viruses either alone or in combination cause mosaic in sugarcane through RT-PCR. Further studies were conducted to detect the associated viruses in a single reaction. To optimize simultaneous detection of these viruses, a new set of primers were designed from the coat protein region of the viruses to suit duplex reverse transcription polymerase chain reaction (D-RT-PCR) and the conditions were standardized to amplify the target viruses in this assay. Of the 29 sugarcane samples tested for the simultaneous occurrence of SCMV and SCSMV, 10 sugarcane varieties were found to be infected with both the viruses with positive amplifications of ∼860 bp and ∼690 bp respectively, for SCMV and SCSMV. In the remaining 19 varieties, SCSMV was detected alone as the causative virus of mosaic. None of the selected varieties were found infected with SCMV alone. Further, the D-RT-PCR was found equally reliable to uniplex RT-PCR performed with SCMV and SCSMV primers in separate reactions. The D-RT-PCR products were cloned, sequenced and the respective sequence information confirmed the authenticity of the viruses amplified in D-RT-PCR. The lengths of the amplified fragments from SCMV isolates were ranged between 812 and 877 bp and in SCSMV it ranged from 683 to 689 bp. This diagnostic approach will be useful to establish distribution map of the virus(es) associated with mosaic in India and in other sugarcane growing countries.

Rapid and Simultaneous Detection of Avian Influenza and Newcastle Disease Viruses by Duplex Polymerase Chain Reaction Assay

A duplex reverse transcription-polymerase chain reaction (dRT-PCR) assay has been developed for the simultaneous, rapid and specific detection/discrimination of avian influenza virus (AIV) and Newcastle disease virus (NDV). Primers targeting the matrix protein gene (M) of AIV and the fusion protein gene (F) of NDV were evaluated experimentally with 13 AIV and 19 NDV strains. PCR products of the expected size of 144 bp and 316 bp were amplified from AIV/NDV samples, respectively, while no cross-reaction was observed with negative controls or with 16 other avian pathogens. The endpoint of detection was defined as approximately 10(+0.5) 50% egg infectious dose (EID(50))/0.2 ml for AIV and 10(+2.2) EID(50)/0.2 ml for NDV. The assay was able to detect AIV/NDV with similar sensitivity in spiked stool samples and in specimens from vaccinated birds. The developed dRT-PCR assay is a rapid, cost-effective tool, which provides powerful novel means for the early diagnosis of avian influenza and Newcastle disease.

Impact of coccidial infection on vaccine- and vvIBDV in lymphoid tissues of SPF chickens as detected by RT-PCR

BackgroundThis study aimed at investigating a potential effect caused by coccidia on the immune response to vaccine- and very virulent infectious bursal disase virus (vvIBDV) in SPF chickens.MethodsTwo groups of three weeks old SPF chickens were vaccinated prior to inoculation with coccidia and challenge with virulent IBDV, all within a period of eight days. Two control groups were similarly treated, except that challenge with field virus was omitted in one group while inoculation with coccidia was omitted in the other group. Clinical signs, lesions in the intestines caused by coccidia, lesions in the bursa of Fabricius caused by IBDV, IBDV-antibody titres, and virus detection by reverse transcription polymerase chain reaction (RT-PCR) were compared among the groups. Lymphoid tissues and swab samples were analysed by general RT-PCR, and positive results were identified by strain specific duplex (DPX) RT-PCR.ResultsIn the tripple-infected groups, vaccine strain IBDV was detected in spleen and thymus tissues, and no field virus was detected in bursa samples, contrary to the double-infected groups.ConclusionThe results suggest an enhancing effect on the immune response caused by subclinical coccidiosis and vvIBDV acting in concert.

Molecular analysis of the RET and NTRK1 gene rearrangements in papillary thyroid carcinoma in the Polish population

Among different genetic factors involved in the pathogenesis of the papillary thyroid carcinoma (PTC), rearrangements of RET protooncogene ( RET/PTC), as well as rearrangements of NTRK1 protooncogene are best known. The resulting hybrid oncogenes are found in PTCs with variable frequency, depending on the examined population. The relationship between these chromosomal aberrations and clinical outcome of PTCs remains still controversial. The study aimed at estimating the frequency of rearrangements of RET and/or NTRK1 protooncogenes in PTC in the Polish population, and at evaluating the possible relationships between the presence of RET and/or NTRK1 oncogenes and such parameters, as patient's age, gender, histopathological variant of tumor and clinical staging. Expression analysis of RET and NTRK1 was performed by duplex reverse transcription-polymerase chain reaction (duplex RT-PCR) and OneStep RT-PCR, respectively, in tumor tissues obtained from 33 patients with PTC. Rearrangements of the RET protooncogene ( RET/PTC1, RET/PTC2 and RET/PTC3) were detected in 7 out of 33 PTC (21%), and rearrangements of NTRK1 [ Trk–T1 and Trk( TPM3)] were detected in 4 out of 33 examined samples (12%). In none of the examined cases, did the RET and NTRK1 rearrangements occur in the same sample. No correlations were found between RET/PTC or Trk oncogenic sequences and patient's age, gender, the histopathological variant of PTC and the assignment to particular stage in clinical staging systems (TNM Staging, the University of Chicago clinical class, and Ohio State University Staging). Our study is the first one in which the frequency of NTRK1 rearrangements in PTC was reported for the Polish population. On the other hand, the frequency of RET rearrangements in PTC, as found by us, was similar to the previously reported results for the Polish population. Our results do not confirm the relationship between the structural aberrations in question and the clinical outcome of PTC.

Serological and molecular detection of tobacco veinal necrosis isolates ofPotato virus Y (PVYN) from potatoes grown in the Western United States

Isolates ofPotato virus Y potyvirus that induced systemic veinal necrosis in tobacco (PVYN) were transmitted from potatoes grown in Washington, Oregon, California, and Idaho. The detection of these viruses by serological and molecular methods was evaluated. In addition to PVYN-like isolates, putative recombinant isolates (PVYN:O) were detected. ELISA tests using monoclonal antibodies specific for ordinary (PVYO) or necrosis-inducing (PVYN) serotypes of the virus indicated that both O and N serotypes were represented among the PVYN isolates. All of the necrosis-inducing PVYN and PVYN:O isolates were rapidly detected and differentiated from the ordinary strain using a novel coupled, one-tube, duplex reverse transcription-polymerase chain reaction (cRT-PCR) assay.

Detection of vvIBDV in Vaccinated SPF Chickens

The purpose of our experiment was to investigate, if apparently healthy, vaccinated chickens may be involved in maintaining and spreading infectious bursal disease virus (IBDV) in poultry environments. We aimed at simultaneous detection and identification of very virulent field strain IBDV (vvIBDV) as well as vaccine strain IBDV in experimentally infected chickens. Two groups of specific pathogen free (SPF) chickens were vaccinated using the intermediate infectious bursal disease (IBD) vaccine D78. Group 1 was vaccinated at the age of one week and group 2 at the age of three weeks. Both groups were challenged with vvIBDV at the age of four weeks. A third, vaccinated, non-challenged group served as negative control. No clinical symptoms were observed in any of these groups. The chickens were euthanised and submitted to autopsy and sample preparation in groups of three at fixed intervals from the age of 28 to 44 days. Gross pathological lesions were not observed. Lymphoid tissues from the bursa of Fabricius, bone marrow, spleen and thymus in addition to cloacal- and bursal swaps were analysed by one-step reverse transcription polymerase chain reaction (RT-PCR). Positive results were confirmed by two-step strain specific duplex (DPX) RT-PCR. The vaccine strain was detected in bursa tissues from all groups, while the challenge strain was detected in few bursal as well as non-bursal tissue samples.The results indicate a possibility of replication of vvIBDV in vaccinated chickens.

Quantification of RNA degradation by semi-quantitative duplex and competitive RT-PCR: a possible indicator of the age of bloodstains?

In vitro RNA degradation is a complex and non-linear process which can serve as indicator for the quality and age of stains. We have developed a semi-quantitative duplex reverse transcription-polymerase chain reaction (RT-PCR) assay which, in combination with competitive RT-PCR using an external standard, allows quantification of RNA degradation levels. Using this method, we have investigated 106 bloodstains stored up to 15 years. The distribution of the peak area quotients of standard and target messenger-RNA (mRNA) as measured by laser-induced fluorescence capillary electrophoresis was closely correlated with the age of the samples. Further statistical analysis showed that bloodstains with age differences of 5 years and more exhibit statistical significant variances in peak area quotients of both housekeeping genes included in this study, β-actin and cyclophilin. This can be of value when evidence from old cases is re-investigated. Our data show, that, although RNA continues to be degraded in dried bloodstains, mRNA suitable for RT-PCR can be isolated from samples stored for at least 15 years.

Detecting Rice stripe virus (RSV) in the small brown planthopper ( Laodelphaxstriatellus) with high specificity by RT-PCR

Rice stripe disease, caused by Rice stripe virus (RSV), may lead to severe or even crippling losses in many rice-cultured countries and regions. As the most important vector of RSV, the small brown planthopper (SBPH) ( Laodelphax striatellus) is largely responsible for the epidemic phase of the disease. Therefore, a rapid identification of RSV in the SBPH is of a great need for disease forecasting. A reverse transcription polymerase chain reaction (RT-PCR) assay is described to amplify a RSV gene in individual L. striatellus. By using primers matched to the viral RNA dependent RNA polymerase gene in RNA1, a 445 bp product was detected in viruliferous SBPHs. Meanwhile, the PCR products produced by the SBPH actin primers constructed across the boundary of an intron and an exon were used as RNA specific positive control for each stage of the experiment to ensure the validity of the negative results. Duplex RT-PCR conditions were established for the simultaneous detection of RSV and actin. This approach can be used for the early detection of RSV in L. striatellus and the subsequent rice stripe disease forecasting.

Possible Escape of a Recombinant Isolate of Potato virus Y by Serological Indexing and Methods of its Detection.

Surveys of commercial and seed potato fields for virus diseases (1998 to 2002) in Manitoba established that Potato virus Y (PVY) is of concern in seed potato production. To determine the prevalence of PVY strains, PVY-infected tubers identified by reverse transcription-polymerase chain reaction (RT-PCR) from surveys (2000 to 2001) were grown for symptom expression and strain characterization by strain-specific RT-PCR, bioassays, and serological assays. Of the samples collected (2000 to 2001) and tested by RT-PCR, 4.0% contained PVY. Further analysis of the PVY-positive samples by a duplex RT-PCR facilitating the simultaneous detection of common (PVYO) and tobacco veinal necrosis strains (PVYN/NTN) indicated that 37.5% contained PVYO and 63.5% contained PVYN-type isolates. Analysis of the PVYN-type samples using three monoclonal antibodies (MAbs) showed that all reacted with only the PVYO MAbs and not with the PVYN-specific MAb. Partial nucleotide sequences of both ends of PVY-RNA showed that the PVYN-type isolates resembled those reported in 1996 from Manitoba. These isolates are designated as PVYN:O. In view of the increased incidence of PVYN:O in one production area, seed tubers imported from other provinces of Canada and the neighboring United States were analyzed for PVYN:O. The PVYN:O was detected in imported seeds from Minnesota, Montana, and North Dakota.