Single-step Reverse Transcription-polymerase Chain Reaction Research Articles

Novel Canine Coronavirus Isolated from a Hospitalized Patient With Pneumonia in East Malaysia.

BackgroundDuring the validation of a highly sensitive pan-species coronavirus (CoV) semi-nested RT-PCR assay, we found canine CoV (CCoV) RNA in nasopharyngeal swabs from eight (2.5%) of 301 patients hospitalized with pneumonia during 2017-18 in Sarawak, Malaysia. Most patients were children living in rural areas with frequent exposure to domesticated animals and wildlife.MethodsSpecimens were further studied with universal and species-specific CoV and CCoV one-step RT-PCR assays, and viral isolation was performed in A72 canine cells. Complete genome sequencing was conducted using Sanger method.ResultsTwo of eight specimens contained sufficient amounts of CCoVs as confirmed by less-sensitive single-step RT-PCR assays, and one specimen demonstrated cytopathic effects (CPE) in A72 cells. Complete genome sequencing of the virus causing CPE identified it as a novel canine-feline recombinant alphacoronavirus (genotype II) that we named CCoV-HuPn-2018. Most of CCoV-HuPn-2018 genome is more closely related to a CCoV TN-449, while its S gene shared significantly higher sequence identity with CCoV-UCD-1 (S1 domain) and a feline CoV WSU 79-1683 (S2 domain). CCoV-HuPn-2018 is unique for a 36 nt (12-aa) deletion in the N protein and the presence of full-length and truncated 7b non-structural protein which may have clinical relevance.ConclusionsThis is the first report of a novel canine-feline recombinant alphacoronavirus isolated from a human pneumonia patient. If confirmed as a pathogen, it may represent the eighth unique coronavirus known to cause disease in humans. Our findings underscore the public health threat of animal CoVs and a need to conduct better surveillance for them.

Detection of coronaviruses in Pteropus & Rousettus species of bats from different States of India.

Background & objectives:Bats are considered to be the natural reservoir for many viruses, of which some are potential human pathogens. In India, an association of Pteropus medius bats with the Nipah virus was reported in the past. It is suspected that the recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) also has its association with bats. To assess the presence of CoVs in bats, we performed identification and characterization of bat CoV (BtCoV) in P. medius and Rousettus species from representative States in India, collected during 2018 and 2019.Methods:Representative rectal swab (RS) and throat swab specimens of Pteropus and Rousettus spp. bats were screened for CoVs using a pan-CoV reverse transcription-polymerase chain reaction (RT-PCR) targeting the RNA-dependent RNA polymerase (RdRp) gene. A single-step RT-PCR was performed on the RNA extracted from the bat specimens. Next-generation sequencing (NGS) was performed on a few representative bat specimens that were tested positive. Phylogenetic analysis was carried out on the partial sequences of RdRp gene sequences retrieved from both the bat species and complete viral genomes recovered from Rousettus spp.Results:Bat samples from the seven States were screened, and the RS specimens of eight Rousettus spp. and 21 Pteropus spp. were found positive for CoV RdRp gene. Among these, by Sanger sequencing, partial RdRp sequences could be retrieved from three Rousettus and eight Pteropus bat specimens. Phylogenetic analysis of the partial RdRp region demonstrated distinct subclustering of the BtCoV sequences retrieved from these Rousettus and Pteropus spp. bats. NGS led to the recovery of four sequences covering approximately 94.3 per cent of the whole genome of the BtCoVs from Rousettus bats. Three BtCoV sequences had 93.69 per cent identity to CoV BtRt-BetaCoV/GX2018. The fourth BtCoV sequence was 96.8 per cent identical to BtCoV HKU9-1.Interpretation & conclusions:This study was a step towards understanding the CoV circulation in Indian bats. Detection of potentially pathogenic CoVs in Indian bats stresses the need for enhanced screening for novel viruses in them. One Health approach with collaborative activities by the animal health and human health sectors in these surveillance activities shall be of use to public health. This would help in the development of diagnostic assays for novel viruses with outbreak potential and be useful in disease interventions. Proactive surveillance remains crucial for identifying the emerging novel viruses with epidemic potential and measures for risk mitigation.

Retrospective study of hepatitis c virus relapse after treatment with sofosbuvir and daclatasvir with or without ribavirin

Background and aims The highest prevalence of chronic hepatitis C virus (HCV) genotype 4 infection is reported in Egypt. Fortunately, the oral anti-HCV therapy (OAT) has been available with more than 95% reported 12-week sustained virologic response (SVR) after treatment for 84 days. The current study goals included evaluation of 12-week SVR after treatment with sofosbuvir (SOF) plus daclatasvir (DCV) with or without ribavirin for 12 weeks. An additional goal was to study the correlation between post-OAT HCV serologic relapse and diabetes mellitus, liver fibrosis, and pretreatment viral loads. Patients and methods Patients with chronic HCV infection (n=590) were retrospectively enrolled in the current study. They were subjected to OAT for 12 weeks. Single daily dose of SOF (400 mg) plus DCV (60 mg) constituted the baseline therapy in all study cases, and ribavirin (RBV) was coadministered in 262 patients. Responses to OAT were assessed 12 weeks after end of treatment by single-step reverse transcription polymerase chain reaction (SRT-PCR). Results We found that overall 12-week SVR was 98% (579/590), which showed insignificant advantages on adding ribavirin (99%) compared with solitary directly acting antivirals regimen (97%) (P>0.05). The SVR in normoglycemic and/or none or early hepatic fibrosis (F0–F2) (>99.0%) was significantly higher compared with diabetic and/or late fibrotic (F3–F4) (94%) patients (P 0.05). Conclusion SOF plus DCV with or without ribavirin regimens achieved 12-week SVR in 98% of the patients with chronic HCV infection. Hepatic fibrosis and diabetes mellitus have negative effects on 12-week SVR. Despite F4 hepatic fibrosis being associated with the highest pre-treatment HCV-SRT-PCR values, baseline viral loads do not affect anti-HCV oral therapy outcomes.

Palm-Sized Device for Point-of-Care Ebola Detection.

We show the utilization of a recently developed cellphone-sized real-time polymerase chain reaction (PCR) device to detect Ebola virus RNA using single-step reverse transcription PCR (RT-PCR). The device was shown to concurrently perform four PCRs, each with a sample volume of 100 nL: one positive control with both Ebola and GAPDH RNA and one negative control. The last two positions were used to measure the GAPDH and the Ebola content of a sample. A comparison of threshold cycles (CT) from the two samples provided relative quantification. The entire process, which consisted of reverse transcription, PCR amplification, and melting curve analysis (MCA), was conducted in less than 37 min. The next step will be integration with a sample preparation unit to form an integrated sample-to-answer system for point-of-care infectious disease diagnostics.

Identification of Peanut bud necrosis virus and Tomato spotted wilt virus in Indonesia for the first time

In surveys conducted in May 2008 in Warung Kondang, Cianjur, West Java, tomato plants ( Solanum lycopersicum ) growing in farmers’ fields showed stunting and leaf symptoms of either bronzing or general chlorosis with vein-banding. In addition, chilli peppers ( Capsicum spp.) were observed with chlorosis and vein-banding symptoms in Salabintana, Sukabumi, West Java. Finally, peanut plants ( Arachis hypogaea ) showing chlorotic rings and necrosis on leaves were observed in a field near to the Bogor Agricultural University Campus, Darmaga Bogor. All these symptoms suggested the possible involvement of tospoviruses. To identify the viruses involved, leaves with symptoms were pressed onto FTA classic cards [FTA ® Classic Card, Whatman International Ltd.] and shipped to Washington State University, with the appropriate permit from USDA-APHIS-PPQ. Nucleic acids were recovered from the cards using a sample extraction protocol (Alabi et al ., 2008) and tested by one-tube, single-step reverse transcriptionpolymerase chain reaction (RT-PCR) using degenerate ‘universal’ tospovirus primers [Upstream primer: 5 ′ -CCTTTAACAGT(A/T/G)GAAACAT-3 ′ and Downstream primer: 5 ′ -CAT(A/T/G)GC(A/G)CAAGA(A/G)TG(A/G)TA(A/ G)ACAGA-3 ′ ] conserved in the L RNA segment of tospoviruses (Chu et al ., 2001). A DNA fragment of approximately 800 base pairs was amplified from the tomato, pepper and peanut samples. Each was cloned separately into pCR 2.1-TOPO vector (Invitrogen). Three independent clones from each sample were sequenced in both directions. A comparison of the nucleotide sequences obtained from the tomato (GenBank Accession No. FJ 177301) and chilli pepper samples (FJ 177302) was made with corresponding sequences of tospovirus L-RNA deposited in GenBank. This analysis showed 96 and 97% nucleotide sequence identity with Tomato spotted wilt virus (TSWV) isolates from Hawaii, USA (AY070218) and Japan (AB198742), respectively. The sequences obtained from tomato and chilli shared only 93% identity between them, indicating that the Indonesian TSWV isolates from tomato and pepper may be distinct strains. An analysis of the nucleotide sequence obtained from the peanut sample (FJ177300) showed 94% sequence identity with corresponding L RNA sequence of a Peanut bud necrosis virus (PBNV) isolate from India (AF025538). These results confirm for the first time that two distinct tospoviruses, TSWV and PBNV, occur in Indonesia.

A simple and rapid single-step multiplex RT-PCR to detect Norovirus, Astrovirus and Adenovirus in clinical stool samples

A single-step multiplex reverse transcription-polymerase chain reaction (RT-PCR) assay that detects and identifies Norovirus, Astrovirus and Adenovirus in clinical stool samples is described. Four hundred sixty stool samples were tested from patients with non-rotavirus acute gastroenteritis, that were either stored at −80 °C and tested retrospectively, or tested immediately after viral nucleic acid extraction in a prospective manner, including outbreaks of gastroenteritis that occurred in Germany during the winter of 2003. The multiplex RT-PCR was validated against simplex RT-PCR with published primers for Norovirus (JV12/JV13 and p289/p290) and Astrovirus (Mon340/348), and against simplex PCR for Adenovirus. In both retrospective and prospective settings, the multiplex RT-PCR was equally sensitive and specific in detecting non-rotavirus infections compared with simplex RT-PCR/PCR. The specificity of the multiplex RT-PCR was assessed by sequencing of the amplicons that showed high nucleotide identities to Norovirus genogroup I/1, I/4, II/2, or II/4 clades, as well as to Astrovirus serotypes 1, 2, 4, or 8. The multiplex RT-PCR was also more sensitive than Astrovirus and Norovirus antigen enzyme immunoassays (IDEIA, Dako), as well as Astrovirus isolation in cell culture. This novel multiplex RT-PCR is an attractive technique for the rapid, specific, and cost-effective laboratory diagnosis of non-rotavirus acute gastroenteritis.

A simple and rapid single-step multiplex RT-PCR to detect Norovirus, Astrovirus and Adenovirus in clinical stool samples

A single-step multiplex reverse transcription-polymerase chain reaction (RT-PCR) assay that detects and identifies Norovirus, Astrovirus and Adenovirus in clinical stool samples is described. Four hundred sixty stool samples were tested from patients with non-rotavirus acute gastroenteritis, that were either stored at −80 °C and tested retrospectively, or tested immediately after viral nucleic acid extraction in a prospective manner, including outbreaks of gastroenteritis that occurred in Germany during the winter of 2003. The multiplex RT-PCR was validated against simplex RT-PCR with published primers for Norovirus (JV12/JV13 and p289/p290) and Astrovirus (Mon340/348), and against simplex PCR for Adenovirus. In both retrospective and prospective settings, the multiplex RT-PCR was equally sensitive and specific in detecting non-rotavirus infections compared with simplex RT-PCR/PCR. The specificity of the multiplex RT-PCR was assessed by sequencing of the amplicons that showed high nucleotide identities to Norovirus genogroup I/1, I/4, II/2, or II/4 clades, as well as to Astrovirus serotypes 1, 2, 4, or 8. The multiplex RT-PCR was also more sensitive than Astrovirus and Norovirus antigen enzyme immunoassays (IDEIA, Dako), as well as Astrovirus isolation in cell culture. This novel multiplex RT-PCR is an attractive technique for the rapid, specific, and cost-effective laboratory diagnosis of non-rotavirus acute gastroenteritis.

Dengue virus infection rate in field populations of female Aedes aegypti and Aedes albopictus in Singapore.

We developed a single-step reverse transcription-polymerase chain reaction (RT-PCR) followed by a semi-nested PCR using an upstream consensus primer and four type-specific primers within the non-structural protein gene (NS3) of dengue viruses to type dengue viruses in field populations of female Aedes mosquitoes. This yielded diagnostic fragments of 169, 362, 265 and 426 base pairs for dengue virus types 1, 2, 3 and 4, respectively. From 1997 to 2000, 54 (6.9%) of 781 Aedes aegypti and 67 (2.9%) of 2256 Aedes albopictus screened were positive for dengue viruses, with a declining trend. The most common dengue virus type detected in the Aedes mosquitoes was dengue-1. Details on the change of one serotype to another in the mosquito population over three consecutive years are discussed.

Influenza virus types and subtypes detection by single step single tube multiplex reverse transcription-polymerase chain reaction (RT-PCR) and agarose gel electrophoresis

Influenza virus type and subtype specific primers were selected for use in reverse transcription polymerase chain reaction (RT-PCR). The selected primer sets were used in a single step RT-PCR of influenza virus RNA in multiplex format for the detection of virus type and subtypes. Three one step reaction conditions are optimized: (1) multiplex typing only, (2) multiplex subtyping of influenza A, and (3) multiplex typing and subtyping simultaneously. RNA from strains of influenza virus type A of subtypes H1N1, H3N2 and H5N1 and influenza virus type B was used for amplification and detection. The amplified DNA fragments were size analyzed for the presence of specific type and subtypes target DNA by agarose gel electrophoresis. The sensitivity of detection was about 0.01 TCID 50 for both influenza type A and B when amplification was for typing alone. In multiplex subtyping or multiplex typing and subtyping simultaneously, the sensitivities for types and subtypes specific bands were 0.01–0.1 TCID 50 in the tested volume.

Single-step reverse transcription-polymerase chain reaction for the detection of hepatitis C virus RNA.

We developed a novel single-step reverse transcription-polymerase chain reaction (RT-PCR), which is equal in sensitivity and specificity to RT-nested PCR, based on both reverse transcriptase and Taq DNA polymerase working efficiently under single buffer reaction conditions. Using in vitro synthesized hepatitis C virus (HCV) RNA, it was demonstrated that 10-100 copies of HCV RNA could be detected with a set of primers that amplify a 144 base-pair sequence unique to the 5'-noncoding region of HCV RNA. Furthermore, this method was successfully performed on serum and liver biopsy specimens obtained from patients with chronic hepatitis C. In addition, HCV RNA from in vitro HCV-infected MT-2C cells, which supported HCV replication, was also detected by this method. The method is anticipated to improve the detection of small amounts of RNA, such as that of HCV, promoting both labor savings and the prevention of carry-over contamination.

Comparison of competitive and non-competitive reverse transcription-polymerase chain reaction (RT-PCR) for the quantification of hepatitis C virus (HCV) RNA

A competitive reverse transcription (RT)-nested polymerase chain reaction (PCR) assay for HCV RNA was compared with the Roche Amplicor HCV Monitor assay, based on non-competitive, single step RT-PCR. A total of 83 serum samples were tested in parallel by both assays. All samples could be quantified by competitive RT-PCR (cPCR), whereas seven were negative by the non-competitive assay (ncPCR). The HCV RNA titre of the discordant samples assessed by cPCR was significantly lower than that of the remaining 76 ( P < 0.001). Absolute HCV RNA titres were higher by cPCR than by ncPCR ( P < 0.001), even if the results of the two methods were statistically correlated ( P < 0.001). HCV RNA titre tested by cPCR was not significantly different between samples infected with genotype 1 or 2. However, values obtained by ncPCR were higher in samples with genotype 1 ( P < 0.001). Furthermore, all seven discordant samples were infected with genotype 2. When both methods were used to measure serial dilutions of standard HCV RNA, we observed a bias to lower measurements with the ncPCR kit. This study shows a good correlation between the results of two PCR-based methods for the quantification HCV RNA. However, the degree of sensitivity and the absolute HCV RNA titre measured may vary according to the assay used.

A rapid and highly specific technique to detect hepatitis C RNA in frozen sections of liver.

To develop a polymerase chain reaction (PCR) based technique that would permit the rapid and highly specific detection of hepatitis C virus (HCV) RNA extracted from frozen liver tissue. Samples of liver tissue from 18 patients undergoing orthotopic liver transplantation were studied. Nine patients were HCV positive. Total RNA was extracted from between one and 10 sections, 10 microns thick, from each tissue sample. HCV RNA was amplified by (1) conventional, multistep reverse transcription PCR (RT-PCR) and by (2) combined, single step RT-PCR using coupled oligonucleotide primers based on the sequence of the 5' untranslated region of the viral genome. Positive results were confirmed by dot blot analysis using a digoxigenin labelled oligoprobe (Alx 89). HCV RNA was detected in the nine HCV positive patients by both conventional and combined RT-PCR. HCV RNA was not detected in the HCV negative patients. As little as 500 ng total RNA was needed as the template to yield detectable amounts of amplified cDNA. The digoxigenin labelled oligoprobe hybridised with HCV RNA positive specimens only. The combined, single step RT-PCR is a rapid and sensitive technique for detecting HCV RNA in frozen liver tissue.

Detection of hepatitis C virus RNA by a reliable, optimized single-step reverse transcription polymerase chain reaction

A single-step reverse transcription polymerase chain reaction (SRT-PCR) method was optimized for hepatitis C virus (HCV) RNA detection. Extraction procedures by proteinase K and guanidinium isothiocyanate gave similar results. The optimal MgCl2 concentration for the SRT-PCR method was 2 mM with 10 units of superscript M-MLV RNase H-reverse transcriptase and 1 unit of Taq polymerase. Shorter PCR cycling steps gave a 10-fold-increased PCR product compared with longer cycling steps. Twenty-five anti-HCV-positive sera from chronic hepatitis C patients were positive with SRT-PCR, whereas only 17 out of 25 were positive by dissociated RT and PCR (dRT/PCR). Specificity was assessed by twenty negative controls. SRT-PCR was 5-fold more sensitive (5 HCV RNA copies per assay) than dRT/PCR with an HCV RNA transcript. Our SRT-PCR method for HCV RNA detection appears fully adapted for routine use in a medical virology laboratory.

Semi-nested PCR using NS3 primers for the detection and typing of dengue viruses in clinical serum specimens

Background: More rapid, specific and sensitive tests for the laboratory diagnosis of dengue virus infections are needed. Objective: To develop a semi-nested polymerase chain reaction (PCR) assay based on primers within the NS3 gene for the simultaneous detection and typing of dengue viruses in human sera. Study design: A first round of single-step reverse transcription-polymerase chain reaction (RT-PCR) was carried out with a pair of consensus primers, followed by a second round of semi-nested amplification using the upstream consensus primer and four type-specific down-stream primers. The sensitivity and specificity of the semi-nested PCR assay were determined using plaque- or TCID 50-titrated virus-infected tissue culture fluid, and total RNA extracted from C6 36 cells infected with dengue and other flaviviruses, respectively. A retrospective study was performed on acute sera from thirteen patients with dengue (confirmed by virus isolation) employing semi-nested PCR in parallel with virus re-isolation and a single-step RT-PCR method for the typing of dengue viruses in human sera. Results: The semi-nested PCR assay could detect up to 1 pfu of dengue virus, but not other flaviviruses. The semi-nested PCR and single-step RT-PCR assays correctly typed dengue viruses in twelve and five sera, respectively, whereas none of the sera was positive by virus re-isolation. Conclusions: This semi-nested PCR assay is a sensitive and specific tool for the detection and typing of dengue viruses from viremic human sera.

Single-step reverse transcription-polymerase chain reaction for hepatitis C virus RNA with DNA enzyme immunoassay hybridization

A single reverse transcription-polymerase chain reaction (SRT-PCR) for HCV RNA detection, confirmed by hybridization of amplified products with biotinylated probes using DNA enzyme immunoassay (DEIA), was compared to nested-PCR (N-PCR) on 20 sera from patients with chronic (n = 18) or acute (n = 2) hepatitis. Results obtained by SRT-PCR confirmed by DEIA and by N-PCR identical. All but one of the patients with chronic hepatitis and positive HCV serology as well as the patients with acute hepatitis had detectable HCV RNA in serum; all patients with chronic hepatitis and indeterminate HCV serology and all controls (n = 5) were negative by the two PCR methods. Both SRT-PCR and N-PCR remained positive after 7 x 10(-2) and 5 x 10(-4) dilutions of two HCV RNA-positive sera. The threshold of detection for SRT-PCR was 15 RNA copies per assay, as assessed by testing serial dilutions of an in vitro synthesized 5'-NCR HCV RNA transcript. SRT-PCR confirmed by DEIA for HCV RNA appears to be as sensitive and specific as N-PCR. Furthermore, it is easier to perform, with less of contamination, is less time-consuming, requires fewer enzymes, and it permits semi-quantification of PCR products.