One-step Reverse Transcriptase-polymerase Chain Reaction Research Articles

Rotavirus A Infection Prevalence and Spatio-Temporal Genotype Shift among Under-Five Children in Amhara National Regional State, Ethiopia: A Multi-Center Cross-Sectional Study.

Background: Globally, rotavirus (RV) A (RVA) is the most common cause of severe and sometimes fatal diarrhea in young children. It is also the major cause of acute gastroenteritis among children in Ethiopia. Currently, the WHO has prequalified four RVA vaccines for universal childhood immunization. Ethiopia introduced the monovalent Rotarix vaccine into its national immunization program in 2013. Since then, only a few studies on the burden and genotype distribution of RVA infection post-vaccine introduction have been conducted (mostly at sentinel surveillance sites). Therefore, this study aimed to assess RVA prevalence and genotype distribution among children under five years in Ethiopia (February 2021-December 2022). Methods: This multi-center hospital-based cross-sectional study involved 537 diarrheic children under-five years old. Rotavirus A detection was conducted using a one-step reverse-transcriptase polymerase chain reaction (RT-PCR). Genotyping was conducted by Sanger sequencing of the VP7 (complete) and VP4 (partial) genes. Descriptive analysis and Pearson's chi-squared test were carried out using SPSS version 29. Phylogenetic analysis with 1000 bootstrap replicates was performed using MEGA version 11 software. Statistical significance was set at p < 0.05 for all analyses. Results: The prevalence of RVA infection among diarrheic children was 17.5%. The most prevalent G-types identified were G3 (37%), the previously uncommon G12 (28%), and G1 (20%). The predominant P-types were P[8] (51%), P[6] (29%), and P[4] (14%). The three major G/P combinations observed were G3P[8] (32.8%), G12P[6] (28.4%), and G1P[8] (19.4%). Phylogenetic analysis revealed clustering of Ethiopian strains with the globally reported strains. Many strains exhibited amino acid differences in the VP4 (VP8* domain) and VP7 proteins compared to vaccine strains, potentially affecting virus neutralization. Conclusions: Despite the high RVA vaccination rate, the prevalence of RVA infection remains significant among diarrheic children in Ethiopia. There is an observable shift in circulating RVA genotypes from G1 to G3, alongside the emergence of unusual G/P genotype combinations such as G9P[4]. Many of these circulating RVA strains have shown amino acid substitutions that may allow for neutralization escape. Therefore, further studies are warranted to comprehend the emergence of these unusual RVA strains and the diverse factors influencing the vaccine's diminished effectiveness in developing countries.

Spatial-temporal distribution and sequence diversity of group a human respiratory syncytial viruses in Kenya preceding the emergence of ON1 genotype.

BackgroundHuman respiratory syncytial virus (HRSV) is a major cause of severe viral acute respiratory illness and contributes significantly to severe pneumonia cases in Africa. Little is known about its spatial–temporal distribution as defined by its genetic diversity.MethodsA retrospective study conducted utilizing archived nasopharyngeal specimens from patients attending outpatient clinics in hospitals located in five demographically and climatically distinct regions of Kenya; Coast, Western, Highlands, Eastern and Nairobi. The viral total RNA was extracted and tested using multiplex real time RT‐PCR (reverse transcriptase polymerase chain reaction). A segment of the G‐gene was amplified using one‐step RT‐PCR and sequenced by Sanger di‐deoxy method. Bayesian analysis of phylogeny was utilized and subsequently median joining methods for haplotype network reconstruction.ResultsThree genotypes of HRSVA were detected; GA5 (14.0%), GA2 (33.1%), and NA1 (52.9%). HRSVA prevalence varied by location from 33% to 13.2% in the Highlands and the Eastern regions respectively. The mean nucleotide diversity (Pi[π]) varied by genotype: highest of 0.018 for GA5 and lowest of 0.005 for NA1. A total of 58 haplotypes were identified (GA5 10; GA2 20; NA1 28). These haplotypes were introduced into the population locally by single haplotypes and additional subsidiary seeds amongst the GA2 and the NA1 haplotypes.ConclusionsHRSVA was found across all the regions throughout the study period and comprised three genotypes; GA5, GA2, and NA1 genotypes. The genotypes were disproportionately distributed across the regions with GA5 gradually increasing toward the Western zones and decreasing toward the Eastern zones of the country.

Serological and Molecular Detection of Avian Influenza Virus ‎Hemagglutinin (H5) in Wild Birds in and around Zaria, Nigeria

Avian influenza (AI) has a worldwide distribution and affects domestic and wild birds, thus causing great economic losses to the poultry industry. This study was carried out to detect avian influenza virus H5 antibodies and nucleic acidin some wild birds [Laughing doves (Spilolepia senegalensis), Speckled pigeons (Columba guinea), Cattle egrets (Bubulcus ibis), Senegalese parrots (Poicephalussenegalus), Mallards (Anas platyrhynchos) and Geese (Anseranserini)] in Zaria and its environs, Kaduna State Nigeria. Sera were tested for avian influenza virus (AIV) H5 antibody using competitive enzyme linked immunosorbent assay (cELISA). Pooled oropharyngeal and cloacal swabs of each bird species (8-10 samples) were tested for AIV nucleic acidusing one-stepreverse transcriptase polymerase chain reaction (RT-PCR). Results revealed overall prevalence of 6.62% and 3.85% for AIV antibody and nucleic acid respectively. Based on species, AIV antibody was detected in laughing dove (10%), speckled pigeon (13.64%) and mallard (19.05%).Also, AIV antigen was detected in Senegalese parrot (20%).In conclusion, AIV antibody and antigen were detected in wild birds in Zaria, Nigeria. Thus, these species of birds could play significant roles in the spread of this virus to chickens. Therefore, measures to limit the interactions of these wild birds with chickens should be implemented to minimize the spread of AI.

Microdroplet-based one-step RT-PCR for ultrahigh throughput single-cell multiplex gene expression analysis and rare cell detection

Gene expression analysis of individual cells enables characterization of heterogeneous and rare cell populations, yet widespread implementation of existing single-cell gene analysis techniques has been hindered due to limitations in scale, ease, and cost. Here, we present a novel microdroplet-based, one-step reverse-transcriptase polymerase chain reaction (RT-PCR) platform and demonstrate the detection of three targets simultaneously in over 100,000 single cells in a single experiment with a rapid read-out. Our customized reagent cocktail incorporates the bacteriophage T7 gene 2.5 protein to overcome cell lysate-mediated inhibition and allows for one-step RT-PCR of single cells encapsulated in nanoliter droplets. Fluorescent signals indicative of gene expressions are analyzed using a probabilistic deconvolution method to account for ambient RNA and cell doublets and produce single-cell gene signature profiles, as well as predict cell frequencies within heterogeneous samples. We also developed a simulation model to guide experimental design and optimize the accuracy and precision of the assay. Using mixtures of in vitro transcripts and murine cell lines, we demonstrated the detection of single RNA molecules and rare cell populations at a frequency of 0.1%. This low cost, sensitive, and adaptable technique will provide an accessible platform for high throughput single-cell analysis and enable a wide range of research and clinical applications.

Multiplex PCR-based titration (MPBT) assay for determination of infectious titers of the three Sabin strains of live poliovirus vaccine

BackgroundConventional assays to titrate polioviruses usually test serial dilutions inoculated into replicate cell cultures to determine a 50% cytopathic endpoint, a process that is both time-consuming and laborious. Such a method is still used to measure potency of live Oral Poliovirus Vaccine during vaccine development and production and in some clinical trials. However, the conventional method is not suited to identify and titrate virus in the large numbers of fecal samples generated during clinical trials. Determining titers of each of the three Sabin strains co-existing in Oral Poliovirus Vaccine presents an additional challenge.ResultsA new assay using quantitative multiplex polymerase chain reaction as an endpoint instead of cytopathic effect was developed to overcome these limitations. In the multiplex polymerase chain reaction-based titration assay, cell cultures were infected with serial dilutions of test samples, lysed after two-day incubation, and subjected to a quantitative multiplex one-step reverse-transcriptase polymerase chain reaction. All three serotypes of poliovirus were identified in single samples and titers calculated. The multiplex polymerase chain reaction-based titration assay was reproducible, robust and sensitive. Its lower limits of titration for three Sabin strains were 1–5 cell culture 50% infectious doses per ml. We prepared different combinations of three Sabin strains and compared titers obtained with conventional and multiplex polymerase chain reaction-based titration assays. Results of the two assays correlated well and showed similar results and sensitivity. Multiplex polymerase chain reaction-based titration assay was completed in two to 3 days instead of 10 days for the conventional assay.ConclusionsThe multiplex polymerase chain reaction-based titration (MPBT) is the first quantitative assay that identifies and titrates each of several different infectious viruses simultaneously in a mixture. It is suitable to identify and titrate polioviruses rapidly during the vaccine manufacturing process as a quality control test, in large clinical trials of vaccines, and for environmental surveillance of polioviruses. The MPBT assay can be automated for high-throughput implementation and applied for other viruses including those with no cytopathic effect.

Antigenic site of nucleoprotein gene from Indonesian rabies virus isolates.

Background and Aim:Several molecular studies on rabies virus (RABV) have been conducted in Indonesia, but it does not give clear information about molecular characteristics of previous RABV isolate in Indonesia. This study was conducted to know the characteristic of circulating RABV to determine a suitable method to control the spreading of RABV in Indonesia.Materials and Methods:Samples of infected RABV from dog brain were collected from Sumatera, Kalimantan, Sulawesi, and Bali Islands. All samples were examined based on nucleoprotein encoding gene to determine the molecular characteristics based on homology and phylogenetic tree compared to Pasteur Virus and RABV that came from another country within Asia (Indonesia, China, Thailand, India, and Korea). The collected samples were processed by one-step reverse transcriptase-polymerase chain reaction using nucleoprotein encoding gene followed by sequencing. The amino acid of its antigenic site of isolated RABV was also analyzed.Results:The results showed that isolated RABV has 84-85% similarity compared to Pasteur. According to phylogenetic construction, isolated samples do not share the same lineage toward Pasteur. The homology scores of isolated samples compared to RABV within Asia such as Indonesia, China, Thailand, India, and Korea were 98-99%, 92-93%, 88-89%, 86-88%, and 85-88%, respectively. According to antigenic site analysis compared to Pasteur, it was found that there were amino acid mutations within antigenic Site IV of nucleoprotein. Amino acid mutation from isoleucine to valine occurred in amino acid number 240 of 6 Kalimantan, 7 Kalimantan, and 8 Kalimantan. Amino acid mutation from alanine to aspartate and asparagine to threonine occurred within the same antigenic site in amino acid number 246 and 273 of C4 isolate from Sulawesi.Conclusion:According to homology and phylogenetic tree analyses, isolated RABV remained different compared to RABV within Asia and Pasteur. The amino acid mutation occurred in antigenic site of nucleoprotein encoding gene.

Detect the microRNA-203 gene protein expression in mammary gland lesions and their relationships with clinical indicators

Objective To detect the microRNA (miRNA, miR)-203 mRNA expression in invasive ductal carcinoma of the breast (IDC) and adjacent non-cancerous lesions, and their relationships with clinical indicators of IDC and hyperplasia lesions were analyzed. Methods MiR-203 mRNA of 60 breast IDC and 60 normal breast tissues and 99 breast hyperplasia lesions and 22 normal breast tissues were amplified by one-step reverse transcriptase-polymerase chain reaction (RT-PCR) method. Results RT-PCR showed miR-203 mRNA: (1) The standardized coefficient were 1.36, 0.98, 0.79, 0.66 and 0.42, which were gradually lower in normal breast tissues, UDH, ADH, DCIS and IDC (P=0.005). The expression rate and standardized coefficient in IDC were significantly lower than those of breast tissues (P=0.003). (2)The standardized coefficient in cases with axillary node metastasis was significantly lower than that in cases without axillary node matastasis, N0>N1> N2 (P=0.000). (3)The standardized coefficient at stage Ⅰ was significantly higher than that at stage Ⅱ, and stage Ⅱ was significantly higher than stage Ⅲ (P=0.000). (4) The standardized coefficient at grade I was significantly higher than that at grade Ⅱ, and grade Ⅱ was significantly higher than grade Ⅲ (P=0.000). (5)The standardized coefficient at estrogen receptor (ER), progesterone receptor (PR), C-erbB-2 (-) was significantly higher than ER, PR, C-erbB-2 (+ + ), (+ + + ) (P=0.003). Conclusion The miR-203 protein expression in IDC and hyperplasia lesions have different levels. miR-203 may play a important role in IDC. Key words: MicroRNA-203; Invasive ductal carcinoma of the breast; Breast hyperplasia lesion

A One-Step RT-PCR Array for Detection and Differentiation of Zoonotic Influenza Viruses H5N1, H9N2, and H1N1

Rapid and comprehensive pathogen identification is crucial in zoonotic influenza diagnosis. By optimizing the design of primers and probes and reverse-transcriptase polymerase chain reaction (RT-PCR) conditions, we achieved simultaneous detection of multiple influenza and zoonotic influenza viruses, including H1N1, H5N1, and H9N2 strains, in a one-step, quantitative real-time RT-PCR array (rRT-PCR array) of RNA from multiple influenza strains utilizing a single set of conditions for RT-PCR amplification. The target sequences from all targeted zoonotic influenza viruses were cloned into recombinant RNA virus particles, which were used to evaluate sensitivity, specificity, and reproducibility of the zoonotic influenza viruses RT-PCR array. The detection limit of the array was shown to be between 10(0) and 10(1) copies per reaction, and the standard curve demonstrated a linear range from 10 to 10(6) copies. Thus, the analytical sensitivity of this zoonotic influenza viruses RT-PCR array is 10-100 times higher than conventional RT-PCR. Specificity of the one-step zoonotic influenza viruses RT-PCR array was verified by comparison of results obtained with retroviral-like particles (RVPs), which contained RNA from isolates of seasonal influenza viruses, zoonotic influenza viruses, and other pathogens known to cause acute respiratory disease. The high sensitivity, rapidity, reproducibility, and specificity of this zoonotic influenza viruses rRT-PCR array has been verified as being sufficient to detect the presence of multiple zoonotic influenza viruses in a single assay. The zoonotic influenza viruses RT-PCR array might provide rapid identification of emergent zoonotic influenza viruses strains during influenza outbreaks and disease surveillance initiatives.

Detection of BCR–ABL using one step reverse transcriptase- polymerase chain reaction and microchip electrophoresis

One-step reverse transcriptase polymerase chain reaction (RT-PCR) coupled with microchip electrophoresis (MCE) was established to analyze BCR–ABL fusion gene. The use of one-step RT-PCR could simplify the RT-PCR procedure and thus reduced the risk of contamination and sample consumption. This method also enhanced the sensitivity for amplified target DNA and dramatically shorted the analysis time. Moreover, this assay can simultaneously identify b2a2 and b3a2. Orthogonal array design, which can investigated mutual effects of PCR parameters, was used to optimize the reaction system. This approach was highly effective, reproducible and sensitive, and would be suitable for the determination of BCR–ABL in clinic diagnosis.

Epidemiological Characterization of Respiratory Viruses Detected from Acute Respiratory Patients in Seoul

Background: Viruses that cause acute respiratory infection include adenovirus (ADV), respiratory syncytial virus A and B (RSV(A), RSV(B)), influenza virus A and B (FluA, FluB), parainfluenza virus 1, 2 and 3 (PIV1, PIV2, PIV3), human metapneumovirus (hMPV), human coronavirus (hCoV), human rhinovirus (hRV), and enterovirus, among others. Methods: Viral incidence was evaluated in acute respiratory patients in Seoul, Korea from 2010 to 2012. A total of 2,544 oropharyngeal swab specimens were tested by real-time polymerase chain reaction (PCR) methods. RNA viruses (hRV, PIV, RSV, hCoV, and hMPV) and DNA viruses (ADV and bocavirus) were detected using the one-step reverse transcriptase PCR (RT-PCR) premix kit (SolGent, Korea) from January 2010 to June 2011, and using the real-time PCR kit (Kogenebiotech, Korea) from July 2011 to December 2012. Results: Thirty-two percent (813/2,544) of specimens were positive; 31.9% (294/923) in 2010, 31.4% (232/ 738) in 2011, and 32.5% (287/883) in 2012. The most frequently isolated virus was hRV (40.7%, 331/813), followed by ADV (23.9%, 194/813), RSV (14.1%, 115/ 813), PIV (12.3%, 100/813), and hCoV (8.7%, 71/813). PIV1 was the most commonly isolated PIV, followed by PIV3 and PIV2, respectively. hCo OC43 was the most commonly isolated hCoV, followed by hCoV NL63 and hCoV 229E, respectively. Conclusion: Information on respiratory viruses circulating in Seoul, Korea over the last three years will be helpful in the management of acute respiratory infections, and for larger-scale epidemiological studies. (Ann Clin Microbiol 2013;16:188-195)

Molecular Epidemiology of Human Enterovirus 71 Strains in the Nanchang Region of China in 2011

Hand, foot, and mouth disease (HFMD) is caused by enteroviruses, most commonly enterovirus 71 (EV71) and coxsackievirus A16 (CA16). In general, EV71 infection is more likely to induce severe complications and mortality than other enterovirus infections. The present study focuses on the molecular epidemiology of human EV71 strains in the Nanchang region of China in 2011. Overall, 651 specimens (throat or rectal swabs) were collected, and one-step reverse transcriptase-polymerase chain reaction was performed for analysis. Enteroviruses were detected in 62.2% (405/651) of the specimens. EV71, CA16, and other enteroviruses were found in 292, 73, and 40 specimens, respectively. Phylogenetic analysis of the VP1 region of the 8 EV71 strains found in the Nanchang region indicated that these strains belong to the C4 subgenotype. This study shows that the C4 subgenotype strain of EV71 was prevalent in the HFMD cases of Nanchang in 2011, and it reports the first incidence of adults being infected by EV71 in the Nanchang region. Thus, the surveillance of HFMD epidemiology and monitoring of HFMD severity should be continued.

One-Step Multiplex Reverse-Transcriptase PCR for Detecting Pandemic (H1N1) 2009 Influenza Virus

Pandemic (H1N1) 2009 influenza has spread throughout the world since April 2009 and has caused many human deaths since its first report in humans. Pandemic (H1N1) 2009 influenza virus was first identified in a Canadian pig herd in April 2009 and has been reported in more than ten countries, including Korea. We developed a one-step multiplex reverse transcriptase polymerase chain reaction (RT-PCR) assay based on the matrix gene that discriminates pandemic (H1N1) 2009 influenza virus from endemic swine influenza viruses. The sensitivity of this assay was 100 copies of in vitro-transcribed target RNA and 0.01 tissue culture infective dose (TCID(50)/ml) of virus and was as high as those of conventional influenza A virus common matrix reverse transcriptase PCR assays and real-time reverse transcriptase PCR assays (1 to 200 copies) developed for detecting pandemic (H1N1) 2009 influenza viruses from human and pig samples. This one-step multiplex RT-PCR assay would be a good tool in monitoring pandemic (H1N1) 2009 influenza virus among pig herds on a regular basis.

Lack of Minority K65R-Resistant Viral Populations Detected After Repeated Treatment Interruptions of Tenofovir/Zidovudine and Lamivudine in a Resource-Limited Setting

Lack of Minority K65R-Resistant Viral Populations Detected After Repeated Treatment Interruptions of Tenofovir/Zidovudine and Lamivudine in a Resource-Limited Setting

A Simple Method for Molecular Detection of Swine-Origin and Human-Origin Influenza A Virus

Here we present a real-time one-step reverse transcriptase-polymerase chain reaction SYBR Green assay derived from the method reported by van Elden and collaborators ( 2001 ) that ensures the rapid, sensitive, and cost-effective detection of both seasonal influenza A virus and emerging (H1N1) swine-origin influenza A virus (S-OIV). In addition to this screening test, which successfully detected both seasonal influenza A virus and S-OIV in human clinical samples, we showed that the probe initially designed by van Elden and collaborators could detect seasonal influenza A virus, but not S-OIV; a new probe was designed and tested that specifically detects S-OIV, but not seasonal influenza A. Both probe-based assays were validated by testing human clinical samples and specifically detected either seasonal influenza virus or S-OIV. Finally, in silico analysis of databases predicted that minor modifications of the van Elden primers would facilitate the use of this assay for the broad spectrum detection of all currently characterized variants of influenza A virus, including avian strains.

Real-Time Reverse-Transcriptase–Polymerase Chain Reaction for Salmonella enterica Detection from Jalapeño and Serrano Peppers

Outbreaks of Salmonella linked to fresh produce emphasize the need for rapid detection methods to curb the spread of foodborne pathogens. Reverse-transcriptase-polymerase chain reaction (RT-PCR) detects the presence of mRNA (shorter half-life than DNA), with greater potential of detecting viable pathogens. Real-time RT-PCR eliminates the need for gel electrophoresis and significantly enhances the speed of detection (<1 day) compared with traditional methods (>5 days). The objectives of this research were to apply real-time SYBR Green I-based RT-PCR to detect Salmonella from jalapeño and serrano peppers spiked with low and high inocula of Salmonella. Inoculated and uninoculated peppers were rinsed with water and dried under ultraviolet light for 10 min. Approximately 25 g peppers was inoculated with 10(8) to 10(1) colony forming units (CFU) of Salmonella enterica serovar Typhimurium in a stomacher bag and hand massaged in sterile 0.05 M glycine-0.14 M saline buffer (0.05% Tween, 3% beef extract) for optimal recovery of bacteria. A short preenrichment step of 6 h in buffered peptone water was needed for the detection of low inocula (10(4) CFU/25 g). One-milliliter portions of the extracts were serially diluted, plated on XLT4 agar, and used for RNA extraction with the Qiagen RNeasy Mini Kit. RT-PCR was carried out using SYBR Green I one-step RT-PCR with previously described invA gene primers and an internal amplification control. Detection limits were 10(4) CFU/25 g (approximately 10(2) CFU/g) and 10(7) CFU/25 g (approximately 10(5) CFU/g) Salmonella from enriched and unenriched inoculated peppers, respectively. Even though this method included a 6-h incubation period, the results were still obtainable in 1 day. This method shows promise for applications in routine surveillance and during outbreaks.

Gene expression profile associated with superimposed non‐alcoholic fatty liver disease and hepatic fibrosis in patients with chronic hepatitis C

Hepatic steatosis occurs in 40-70% of patients chronically infected with hepatitis C virus [chronic hepatitis C (CH-C)]. Hepatic steatosis in CH-C is associated with progressive liver disease and a low response rate to antiviral therapy. Gene expression profiles were examined in CH-C patients with and without hepatic steatosis, non-alcoholic steatohepatitis (NASH) and fibrosis. This study included 65 CH-C patients who were not receiving antiviral treatment. Total RNA was extracted from peripheral blood mononuclear cells, quantified and used for one-step reverse transcriptase-polymerase chain reaction to profile 153 mRNAs that were normalized with six 'housekeeping' genes and a reference RNA. Multiple regression and stepwise selection assessed differences in gene expression and the models' performances were evaluated. Models predicting the grade of hepatic steatosis in patients with CH-C genotype 3 involved two genes: SOCS1 and IFITM1, which progressively changed their expression level with the increasing grade of steatosis. On the other hand, models predicting hepatic steatosis in non-genotype 3 patients highlighted MIP-1 cytokine encoding genes: CCL3 and CCL4 as well as IFNAR and PRKRIR. Expression levels of PRKRIR and SMAD3 differentiated patients with and without superimposed NASH only in the non-genotype 3 cohort (area under the receiver operating characteristic curve=0.822, P-value 0.006]. Gene expression signatures related to hepatic fibrosis were not genotype specific. Gene expression might predict moderate to severe hepatic steatosis, NASH and fibrosis in patients with CH-C, providing potential insights into the pathogenesis of hepatic steatosis and fibrosis in these patients.

Surveillance of Emerging Fish Viral Pathogens in Some Southeast Asian Countries

Preventing the transboundary movement of fish viral pathogens in a global environment requires active surveillance. This study examined the presence of three emerging viral pathogens among koi, common, grass, and silver carp in Cambodia, Lao PDR, Myanmar, Philippines, and Vietnam. The stud- ied viruses included koi herpesvirus (KHV), spring viremia of carp virus (SVCV), and grass carp reovirus (GCRV). Detection methods consisted of virus isolation by cell culture, infection assay in naive fish, polymerase chain reaction (PCR), and reverse-transcriptase PCR (RT-PCR). Tissues were collected and pooled from 193 fish samples in Dec. 2004 to Feb. 2005, 406 in Sep. 2005 to Feb. 2006, and 1302 in Oct. 2006 to Feb. 2007. For cell cul- ture, tissue filtrates were prepared from pooled spleens, kidneys, livers, and gills and inoculated onto koi fin (KF-1), grass carp kidney (GCK), and fat head minnow (FHM) cells. For infection assay, tissue filtrates were injected intraperitoneally to healthy, naive common carp. No virus was detected after three cell culture passages and the infection bioassays. One-step and nest- ed-step PCR was used to detect KHV in gills of fish samples. One-step and semi-nested RT-PCR was used to detect SVCV and GCRV in the spleens, kidneys, and livers of fish samples. Samples from all three years from all five countries yielded negative results for all three viruses, indicating that KHV, SVCV, and GCRV were not present in these five countries during the period of the study although KHV outbreaks had been detected in Indonesia, Taiwan, Japan, Thailand, China, and Malaysia.

Genetic diversity of pestivirus isolates in cattle from Western Austria

The genetic diversity of bovine viral diarrhoea virus (BVDV) isolates in infected cattle from Tyrol and Vorarlberg (Austria) was investigated. Blood samples were collected within the compulsory Austrian BVDV control programme during 2005 and 2006. The 5′-untranslated region (5′-UTR) and partially the N-terminal autoprotease (N pro) were amplified by one-step reverse transcriptase-polymerase chain reaction (RT-PCR) and the PCR products were subsequently sequenced. Phylogenetic analysis based on 5′-UTR and N pro sequences demonstrated that almost all isolates (307/310) were of the BVDV-1 genotype. They were clustered into eight different subtypes, here listed by their frequency of occurrence: BVDV-1h (143), BVDV-1f (79), BVDV-1b (41), BVDV-1d (28), BVDV-1e (6), BVDV-1a (4), BVDV-1g (3) and BVDV1-k (3). Two pestivirus isolates were typed as BVDV-2 and one isolate as BDV closely related to Gifhorn strain (BDV-3). Correlation among isolates could only be observed at the farm level, i.e., within a herd. However, no correlation between the genetic and geographical distances could be observed above the farm level. Because of the wide distribution of certain BVDV-1 subtypes and the low prevalence of herd-specific strains, a determination of tracing routes of infection was not possible. Furthermore, recombination events were not detected.

An integrated microfluidic system using magnetic beads for virus detection

An integrated system capable of sample pretreatment using antibody-conjugated magnetic beads and one-step reverse transcriptase–polymerase chain reaction (RT-PCR) on a microfluidic system was developed to accelerate the detection of RNA viruses such as dengue virus or enterovirus 71. The targeted virus in the sample was first captured by the specific antibody-conjugated magnetic beads, which were manipulated by micro-electromagnets made of micro-electro-mechanical systems technology. The RNA of the targeted virus then underwent thermal lysis and was reverse-transcripted to cDNA using a μRT-PCR module. The sensitivity to detect dengue virus is around 10–100 PFU, which is equivalent to the commercial RNA extraction kit and a large-scale RT-PCR machine. This microsystem can specifically detect 4 serotypes of dengue virus, as well as enterovirus 71. The specificity was warranted by both antibody and primer. The microfluidic system allows automatic process of sample including mixing, incubation, and reaction. The antibody-conjugated magnetic beads offer sample pretreatment of purification and concentration. The integration of antibody-conjugated magnetic beads into the microfluidic system is promising for fast molecular diagnosis of microorganisms.

Development of One-Step Reverse Transcriptase–Polymerase Chain Reaction to Detect Duck Hepatitis Virus Type 1

A one-step reverse transcriptase-polymerase chain reaction (RT-PCR) method was developed and optimized for the detection of duck hepatitis virus type 1 (DHV-1) using the Viral Gene-spin viral DNA/RNA extraction kit. A pair of DHV-1-specific primers was designed against the gene encoding RNA-dependent RNA polymerase (3D gene). Using RNA prepared from duckling liver samples infected with two reference and seven Korean field isolates of DHV-1, one-step RT-PCR with DHV1-specific primers amplified a 467-bp fragment. Under the same conditions, no amplification was observed for 14 other avian pathogenic viruses and bacteria. Using RNA prepared from serial dilutions of the DHV-1 with the supernatant of the uninfected duckling liver homogenate (10% w/v), the one-step RT-PCR assay was found to be sensitive to 10 50% egg lethal dose (ELD50) 0.1 ml(-1) of DHV-1. Furthermore, this method detected DHV-1 from the livers and allantoic fluid of duck embryos dying before 3 days postinoculation (PI) and of chicken embryos that were chilled at 3 days PI. Therefore, this one-step RT-PCR method is rapid, sensitive, and reliable, and can be readily adapted for detection of DHV-1 from other clinical samples.