Conventional Reverse Transcription PCR Research Articles

Advancing broad bean true mosaic virus detection using conventional RT-PCR and real-time RT-PCR with novel primer set design

Broad bean true mosaic virus (BBTMV) infects broad beans and peas, reducing yield. As BBTMV is transmitted through broad beans, many countries have implemented regulations to prevent the distribution of infected seeds. Currently, enzyme-linked immunosorbent assay (ELISA) is commonly used to detect BBTMV. While the PCR-based method is preferred for seed virus detection due to its sensitivity and speed. A BBTMV-specific PCR detection method has not yet been reported. A universal detection method currently exists that utilizes reverse transcription PCR (RT-PCR) for the Comovirus genus, to which BBTMV belongs. However, sequence analysis is required for species identification. To address this limitation, we developed and verified RT-PCR detection methods using newly designed BBTMV-specific primers. RT-PCR and real-time RT-PCR with these primers were approximately 5 × 105–106 times more sensitive than ELISA and 100–1000 times more sensitive than previously reported RT-PCR methods. Using RT-PCR and real-time RT-PCR employing these primers, we could detect BBTMV with same sensitivity when more than 3.0 × 105 copies were present per gram of broad bean seeds. Our newly developed detection methods can test for BBTMV with high sensitivity and speed.

Validation of a TaqMan one-step real-time RT-PCR assay targeting ISAV segment 7 spliced mRNA

Infectious salmon anaemia virus (ISAV) can cause severe systemic infection in Atlantic salmon (Salmo salar L.), and a timely diagnosis is critical. Conventional real-time reverse transcription PCR (RT-qPCR) assays target unspliced RNA from either ISAV segment 7 or 8 and provide data on viral load. Here, we evaluate a TaqMan one-step RT-qPCR assay that detects explicitly a spliced messenger RNA (mRNA) of ISAV segment 7, thus providing evidence of active viral transcription. Assay performance was comparable with existing unspliced segment 7 and segment 8 assays. PCR efficiency as evaluated from dilutions of a synthetic DNA fragment was 98 % (R2 = 1.00). The assay also performed well on clinical heart samples with PCR efficiency of 108 % (R2 = 1.00). Finally, evaluation on kidney samples from experimental infection revealed higher levels of active transcription for high-virulent compared to low-virulent ISAV. At early, peak, and late infection, mean ratios of spliced to unspliced segment 7 RNA were 3.0 % (± 0.7), 1.7 % (± 0.3), and 1.5 % (± 0.1) for the low virulent and 9.4 % (± 2.2), 4.7 % (± 0.8), and 6.2 % (± 0.1) for the high virulent isolate, respectively. By detection and quantification of active ISAV transcription, this assay may provide a more detailed understanding of ISAV infection dynamics.

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Detection of Velogenic Avian Paramyxoviruses in Rock Doves in New York City, New York.

ABSTRACTAvian paramyxovirus 1 (APMV-1), also known as Newcastle disease virus (NDV), causes severe and economically important disease in poultry around the globe. Although a limited amount of APMV-1 strains in urban areas have been characterized, the role of the urban wild bird population as an APMV-1 reservoir is unclear. Because urban birds may have an important role for long-term circulation of the virus, fecal and swab samples were collected by community scientists from wild birds in New York City (NYC), New York, United States. These samples were screened for APMV-1 and genotypically characterized by sequencing of the complete genome. A total of 885 samples were collected from NYC parks and from a local wildlife rehabilitation clinic from October 2020 through June 2021, and 255 samples obtained from 197 birds have been processed to date. Eight birds (4.1%) screened positive for the APMV-1 nucleoprotein gene by conventional reverse transcription PCR (RT-PCR), and two live viruses were isolated via egg culture. A multibasic F protein cleavage sequence, 112R R K K R F117, an indicator of highly pathogenic velogenic APMV-1 strains, was present in the two samples fully sequenced by next generation sequencing. Phylogenetic analysis of the F gene coding sequence classified both isolates into genotype VI, a diverse and predominant genotype responsible for APMV-1 outbreaks in pigeon and dove species worldwide.IMPORTANCE Here we describe the first large-scale effort to screen for APMV-1 in New York City’s wild bird population as part of the New York City Virus Hunters program, a community science initiative. We characterized two isolates of APMV-1, with phylogenetic analyses suggesting diversity in established and circulating strains of pigeon paramyxoviruses. Our isolates are also domestic reference strains for future APMV-1 vaccine developments. Future surveillance in this region may contribute to our understanding of APMV-1’s evolution and genetic diversity, as well as inform poultry husbandry and vaccination practices in New York State.

Molecular Characteristics and Incidence of Apple Rubbery Wood Virus 2 and Citrus Virus A Infecting Pear Trees in China.

Apple rubbery wood virus 2 (ARWV-2) and citrus virus A (CiVA) belong to a recently approved family Phenuiviridae in the order Bunyavirales and possess negative-sense single-stranded RNA genomes. In this study, the genome sequence of three ARWV-2 isolates (S17E2, LYC2, and LYXS) and a CiVA isolate (CiVA-P) infecting pear trees grown in China were characterized using high-throughput sequencing combined with conventional reverse-transcription PCR (RT-PCR) assays. The genome-wide nt sequence identities were above 93.6% among the ARWV-2 isolates and above 93% among CiVA isolates. Sequence comparisons showed that sequence diversity occurred in the 5′ untranslated region of the ARWV-2 genome and the intergenic region of the CiVA genome. For the first time, this study revealed that ARWV-2 proteins Ma and Mb displayed a plasmodesma subcellular localization, and the MP of CiVA locates in cell periphery and can interact with the viral NP in bimolecular fluorescence complementation assays. RT-PCR tests disclosed that ARWV-2 widely occurs, while CiVA has a low incidence in pear trees grown in China. This study presents the first complete genome sequences and incidences of ARWV-2 and CiVA from pear trees and the obtained results extend our knowledge of the viral pathogens of pear grown in China.

Molecular detection of H1N1 virus by conventional reverse transcription PCR coupled with nested PCR

Influenza A pdm09 virus has been spreading worldwide and creates a serious public health threat. Among different subtypes/lineages of swine influenza A viruses, the H1N1 subtype is more prevalent in all-over world infections followed by H3N2 which is present in the northern hemisphere. Influenza A pdm09 is dominant for centuries but the last pandemic was in 2009 and is responsible for 151,700–575,400 deaths within one year worldwide (Centres for Disease Control and Prevention). Rapid diagnosis for H1N1 is essential which must be highly sensitive and specific and can differentiate between influenza A subtypes and other respiratory pathogens. In this study, a conventional polymerase chain reaction assay and nested polymerase chain reaction assay were developed which can detect H1N1 pdm09. One outer set of primer is designed and one inner set of primer is designed specifically for hemagglutinin gene (H1). Specificity was checked using different pathogenic genomes for amplification of the H3N2 subtype, Neisseria meningitides, Salmonella typhi, etc. The sensitivity of the nested polymerase chain reaction is 0.001ng/6 ​μL of PCR amplified product of outer primer product of HA1 gene as compared to 50 ​ng/μL of two-step conventional polymerase chain reaction. The nested PCR and conventional PCR are performed on 80 samples which are consistent with the Real-Time PCR results. Real-Time is a standard test for H1N1 detection. But it is very expensive but current method is less expensive and provides comparable results with Real-Time PCR.

Surveillance To Prevent the Spread of Norovirus Outbreak from Asymptomatic Food Handlers during the PyeongChang 2018 Olympics

Human noroviruses are major causes of nonbacterial gastroenteritis and are transmitted by both food and water, as well as person-to-person. Asymptomatic norovirus infection of food handlers may play a role in transmission. The outbreak of norovirus infections was recognized in the PyeongChang Winter Olympics, starting with security staff on 3 February 2018. The Ministry of Food and Drug Safety in the Republic of Korea conducted norovirus surveillance from asymptomatic food handlers of food-catering facilities related to the Olympics to prevent the spread of noroviruses. Rectal swab samples (707) from food handlers were collected and examined for noroviruses by using real-time reverse transcription PCR and conventional reverse transcription PCR. Five of 707 samples were identified as noroviruses. Genotypes of the norovirus-positive samples were determined with sequencing analysis. Identified genotypes of norovirus in asymptomatic food handlers included GI.3, GII.4, and GII.17. The GII.17 strain was prevalent among the genotypes, accounting for three of five detections. Food handlers with noroviruses detected in rectal swabs were excluded from cooking, and all food handled by infected food handlers was discarded. Surveillance of norovirus infection for food handlers contributed to preventing norovirus spread.

Association of Citrus Virus A to Citrus Impietratura Disease Symptoms.

Citrus impietratura disease (CID) is a graft transmissible, virus-like disease observed in old-line citrus trees; its characteristic symptom is the appearance of gum in the albedo of the affected fruits. To identify the causal agent of the disease, high-throughput sequencing (HTS) was performed on symptomatic orange fruits. The analysis of the obtained data revealed in all samples mixed infections of viroids commonly found in citrus trees together with the recently described citrus virus A (CiVA). Examination of additional symptomatic fruits with conventional reverse transcription PCR led to the identification of a single CiVA infection in one tree, which was verified by HTS. Indexing of the single CiVA-infected tree on indicator plants resulted in the appearance of characteristic symptoms in the leaves that were correlated with virus accumulation. Moreover, a comparative analysis among symptomatic and asymptomatic fruits derived from the same trees was performed and included the single CiVA-infected orange tree. The analysis revealed a positive correlation between the appearance of symptoms and the accumulation of CiVA RNAs. To facilitate CiVA detection during certification programs of propagation material, a quantitative RT-PCR targeting the movement protein of the virus was developed and evaluated for reliable and sensitive detection of the virus. To the best of our knowledge this is the first study that associates CiVA with the appearance of CID symptoms.

SARS-CoV-2 Diagnostic Tests: Algorithm and Field Evaluation From the Near Patient Testing to the Automated Diagnostic Platform.

Introduction: Since the first wave of COVID-19 in Europe, new diagnostic tools using antigen detection and rapid molecular techniques have been developed. Our objective was to elaborate a diagnostic algorithm combining antigen rapid diagnostic tests, automated antigen dosing and rapid molecular tests and to assess its performance under routine conditions.Methods: An analytical performance evaluation of four antigen rapid tests, one automated antigen dosing and one molecular point-of-care test was performed on samples sent to our laboratory for a SARS-CoV-2 reverse transcription PCR. We then established a diagnostic algorithm by approaching median viral loads in target populations and evaluated the limit of detection of each test using the PCR cycle threshold values. A field performance evaluation including a clinical validation and a user-friendliness assessment was then conducted on the antigen rapid tests in point-of-care settings (general practitioners and emergency rooms) for outpatients who were symptomatic for <7 days. Automated antigen dosing was trialed for the screening of asymptomatic inpatients.Results: Our diagnostic algorithm proposed to test recently symptomatic patients using rapid antigen tests, asymptomatic patients using automated tests, and patients requiring immediate admission using molecular point-of-care tests. Accordingly, the conventional reverse transcription PCR was kept as a second line tool. In this setting, antigen rapid tests yielded an overall sensitivity of 83.3% (not significantly different between the four assays) while the use of automated antigen dosing would have spared 93.5% of asymptomatic inpatient screening PCRs.Conclusion: Using tests not considered the “gold standard” for COVID-19 diagnosis on well-defined target populations allowed for the optimization of their intrinsic performances, widening the scale of our testing arsenal while sparing molecular resources for more seriously ill patients.

Development of a TaqMan-probe-based multiplex real-time PCR for the simultaneous detection of emerging and reemerging swine coronaviruses

ABSTRACT With the outbreak of the recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019, coronaviruses have become a global research hotspot in the field of virology. Coronaviruses mainly cause respiratory and digestive tract diseases, several coronaviruses are responsible for porcine diarrhea, such as porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and emerging swine acute diarrhea syndrome coronavirus (SADS-CoV). Those viruses have caused huge economic losses and are considered as potential public health threats. Porcine torovirus (PToV) and coronaviruses, sharing similar genomic structure and replication strategy, belong to the same order Nidovirales. Here, we developed a multiplex TaqMan-probe-based real-time PCR for the simultaneous detection of PEDV, PDCoV, PToV, and SADS-CoV for the first time. Specific primers and TaqMan fluorescent probes were designed targeting the ORF1a region of PDEV, PToV, and SADS-CoV and the ORF1b region of PDCoV. The method showed high sensitivity and specificity, with a detection limit of 1 × 102 copies/μL for each pathogen. A total of 101 clinical swine samples with signs of diarrhea were analyzed using this method, and the result showed good consistency with conventional reverse transcription PCR (RT-PCR). This method improves the efficiency for surveillance of these emerging and reemerging swine enteric viruses and can help reduce economic losses to the pig industry, which also benefits animal and public health.

First Report of Grapevine Virus T Infecting Grapevine in China

Grapevine virus T (GVT), a putative new member of the genus Foveavirus in the family Betaflexiviridae, was first described from transcriptomic data of the cultivar Teroldego (Jo et al. 2017). Since then, GVT has been reported from grapevines in Germany (Ruiz-Garcia et al. 2018), Croatia (Voncina et al. 2018), and other European countries (Glasa et al. 2018; Nourinejhad Zarghani et al. 2018). It is closely related to Grapevine rupestris stem pitting-associated virus and exhibited relatively high intraspecies variability. In June 2017, a field survey of grapevine disease in Yanqing district of Beijing was conducted, and grapevine plants with the symptoms of chlorosis and leaf rolling were observed with an incidence of 5 to 40%. Total RNA was extracted from leaf samples of a symptomatic Red Globe grapevine plant and used for constructing a transcriptome library followed by high-throughput sequencing on an Illumina HiSeq 4000 platform. De novo assembly of the 8,290,478 reads was performed by Trinity, and the BLASTn and BLASTX analysis of the assembled 14,834 contigs revealed the presence of one contig related to GVT (8,653 nt in length), with a nucleotide similarity ranging from 81 to 92%, together with the contigs related to Grapevine leafroll-associated virus 1, Grapevine Red Globe virus, and Grapevine Pinot gris virus. Furthermore, the complete genome of this GVT isolate (GVT-BJ) was amplified with conventional reverse transcription PCR (RT-PCR) using the primer pairs F1:5′-TGCTTTCACGTAATGGCTCTTCAC-3′/R1:5′- CCTCAGCAACTCAAAAATGTCAGAAT-3′ and F2:5′-GTGGTGGTCCTTGATGAAATTCAGCT-3′/R2:5′-AGTACGGTATGCCAGCGAACATATGC-3′ designed for the assembled contig and RACE (5′-GATTACGCCAAGCTTCGGGGGTATGAGATTTGCCAATGTTGGC-3′ for 5′ RACE and 5′-GATTACGCCAAGCTTGATGAAAGTGCTTTGGAGCCGGAAGG-3′ for 3′ RACE), and the PCR products were cloned and Sanger sequenced. The genome length of GVT-BJ was 8,700 nt (GenBank no. MH158659) with the typical foveavirus genomic organization with five open reading frames. A pairwise sequence comparison revealed GVT-BJ shared highest nucleotide sequence identity of 92.3% with the French isolate CF-3 (MH674180). Furthermore, 36 symptomatic grapevine samples from nine vineyards in Yanqing district were screened for the presence of GVT by RT-PCR with the primer pair targeting the CP region (CPf 5′-ATGGCGTCAAATGAGGAGC-3′/CPr 5′-TTATGAATCTCCCCCAAATGGATTG-3′); four were positive, and the presence of GVT was further confirmed by sequencing the PCR products. These four grapevine samples were also positive for Grapevine leafroll-associated virus 1 identified by RT-PCR with the reported primers pORF9F and pORF9R (Little and Rezaian 2006). This is the first report of GVT in China in mixed infection with other viruses. A larger-scale survey of commercial vineyards for the prevalence of GVT in China and its association with the observed symptoms is needed.

One-step triplex reverse-transcription PCR detection of porcine epidemic diarrhea virus, porcine sapelovirus, and porcine sapovirus.

Swine diarrhea can be caused by multiple agents, including porcine epidemic diarrhea virus (PEDV), porcine sapelovirus (PSV), and porcine sapovirus (SaV). We designed a one-step triplex reverse-transcription PCR (RT-PCR) detection method including 3 pairs of primers that focused on the S1 gene of PEDV, a conserved gene of PSV, and the VP1 gene of SaV. The optimal concentrations of upstream and downstream primers in the triplex RT-PCR were 0.24 μM for PEDV, 0.15 μM for PSV, and 0.2 μM for SaV, and the optimal annealing temperature was 55.5°C. Triplex RT-PCR assessment of 402 piglet diarrhea samples was compared with conventional individual RT-PCR. Concordance rates in both tests for individual viruses were 100%, 97.6%, and 94.4% for PEDV, PSV, and SaV, respectively. PEDV, PSV, and SaV were detected in 57.2%, 10.4%, and 9.0% of the samples, respectively. The high sensitivity and specificity of this triplex RT-PCR-based detection method for PEDV, PSV, and SaV could allow rapid detection and analysis of mixed infections by these 3 viruses.

Fluorescence resonance energy transfer combined with asymmetric PCR for broad and sensitive detection of porcine reproductive and respiratory syndrome virus 2

With its ever-increasing viral genetic diversity, accurate diagnosis of porcine reproductive and respiratory syndrome virus (PRRSV) infection is indispensable for PRRSV control. Here, a sensitive graphene oxide (GO)-based FRET method was developed to detect PRRSV-2 based on the ability of GO to quench fluorophore by fluorescence resonance energy transfer (FRET). Using primers and a fluorophore-labeled ssDNA probe targeting a conserved region between the PRRSV M gene and 3′UTR, asymmetric PCR specifically amplified viral ssDNA that could anneal with probe to generate dsDNA only in the presence of virus. Upon exonuclease III treatment to release the probe fluorophore, which degrades dsDNA with blunt ends or recessed 3´-termini, the ssDNA annealed with other probe to generate enhanced fluorescence. This GO-based FRET assay specifically detected both classical and highly pathogenic PRRSV, with analytical sensitivity approaching 10 copies/μL, similar to that of real-time PCR but greater than that of conventional reverse transcription PCR (RT-PCR). Consistent with real-time RT-PCR detection, the assay developed here exhibited high diagnostic sensitivity for virus detection of sera from experimentally and naturally infected pigs. Thus, this novel GO-based FRET assay combined with asymmetric PCR detection is sensitive and specific and will be valuable for future PRRSV diagnosis.

Detection and Quantification of Potato virus Y Genomes in Single Aphid Stylets.

Typically, the detection of a plant virus within its vector is carried out on the entire insect body. This process can be a possible source of confusion in the quantification of transmissible virus particles for styletborne viruses such as Potato virus Y (PVY), since the transmissible virus fraction is the one only retained in the aphid vector's mouthparts. The objective of this study was to develop and validate the quantitative PCR method for the detection and quantification of PVY in the vector's stylet. Using a specific method based on TaqMan chemistry with higher sensitivity than conventional reverse transcription PCR, this study reveals that a significant amount of the virus is enclosed within the dissected stylets of Myzus persicae. Because this quantification only concerns the portion of the virus attached to the stylets, uniformity was observed in the recorded numbers of virus targets. This novel assay is applicable to several PVY strains as a rapid and sensitive detection method for use in PVY research and offers a convenient tool for deciphering the mechanism of Potyvirus acquisition.

Molecular epidemiological analysis of Norovirus in patients with foodborne diseases in sentinel hospital, Ma'anshan City, Anhui Province

Objective: To understand the infection status and genetic characteristics of Norovirus from foodborne diseases in sentinel hospital of Ma'anshan city. Methods: The 911 stool samples and epidemiological data of the patients with foodborne disease were collected from three hospitals of Ma'anshan city during January 2015 to June 2018. The G Ⅰ and G Ⅱ Norovirus were detected by real-time reverse transcription PCR. Some of the positive specimens were amplified by conventional reverse transcription PCR, and the PCR products were sequenced for sequence alignment and phylogenetic analysis. Results: The positive rate of Norovirus was 14.7% (134/911), in which 7 strains were G Ⅰ, 124 strains were G Ⅱ and 3 strains were mixed infection. Norovirus can be detected throughout the year, with high positive rate from December to April of the next year [24.4%(20/82)-45.3%(24/53)]. The 78 males (15.5%) and 55 females (13.7%) were positive for Norovirus (χ(2)=0.58,P=0.448). There was no significant difference in different age groups (χ(2)=9.55, P=0.089). A total of 79 strains were successfully sequenced, 4 strains were G Ⅰ group(5.1%), 75 strains were G Ⅱ group (94.9%). The predominant strains were GⅡ.17 and GⅡ.4, aud the number were 35 and 15 respectively. The predominant strains were different in different years. The main strain was GⅡ.17 in 2015 (30, 68.2%),GⅡ.4 in 2016 (5/9) and 2017 (8/16), but GⅡ.3 in 2018 (3/6). Conclusion: Norovirus diarrhea was popular in Ma'anshan city throughout the year,especially in winter and spring. The prevalent strain was GⅡ,genotypes were diversified distribution,the dominant strains were GⅡ.17 and GⅡ.4. The predominant strains were different in different years.

Template-ready PCR method for detection of human telomerase reverse transcriptase mRNA in sputum

Human telomerase reverse transcriptase (hTERT) mRNA in tissue is a biomarker of lung cancer, but hTERT mRNA in sputum had not been successfully detected with conventional reverse transcription PCR methods. Here, we developed a novel PCR protocol: Template-Ready PCR (TRPCR), to detect sputum hTERT mRNA, in which probes serve as templates of amplification. While free probes and dsDNA were removed in template preparation through aspiration and restriction digestion, probes that formed into heterocomplex with target RNA remained intact for PCR amplification. By fishing out the heterocomplex and amplifying the probes, TRPCR achieved sensitivity higher than reverse transcription-quantitative PCR (RT-qPCR). ROC curve of sputum hTERT mRNA by TRPCR assay showed the discrimination in high sensitivity and specificity between patients with lung cancer and lung cancer-free donors at the PCR Ct cutoff of 33. We further validated this approach through TRPCR assay of sputum from 858 lung cancer patients and 480 non-malignant pulmonary disease patients. 722 (84.2%) cases from 858 with lung cancer patients were detected as positive, whereas 461 (96.0%) cases from 480 non-malignant pulmonary disease patients were detected as negative, suggesting that TRPCR assay of sputum hTERT mRNA can serve as a non-invasive molecular diagnosis of lung cancer.

DIAGNOSIS AND PHYLOGENETIC ANALYSIS OF THE CIRCULATING PESTE DES PETITS RUMINANTS VIRUS IN Al-SHARQIA GOVERNATE

Peste des petits ruminants (PPR) is a Morbillivirus within the Paramyxoviridae family which characterized by highly contagious nature with high morbidity and mortality rates in domestic small ruminants. The aim of this study was to investigate an outbreak of PPRV in a flock of sheep and goats in Belbes city, AL-Sharkia governorate in 2018 by virus isolation and conventional RT-PCR. Phylogenetic analysis of N gene sequence of PPRV isolate. Also, compare it with other isolate from previous outbreak in Zagazig city, Al-Sharkia governorate in 2017 as a measurement of the infection status in Egypt. The current study applied on a flock of 55 small ruminants (42 goats and 13 sheep) in Belbes city which suspected to be infected by PPRV. All infected animals were not vaccinated against PPRV and randomly move from place to place. The morbidity rate was 100% and mortality rate was (23.8% in goats and 7.69% in sheep). Diseased animals suffered from fever, mucopurulent ocular discharge, nasal discharge, dyspnea, diarrhea, necrotic tissue and diphtheritic membrane in oral cavity. The ten samples (6 tissue scraping from oral lesions and 4 oculo-nasal swabs) were tested by conventional reverse transcription PCR which revealed 100% sensitivity compared to VI (virus isolation) 70%. Comparative of N gene sequence of both PPRV isolates revealed that homogenous population of PPR virus isolates up to 99%. Also, the PPRV isolate of the current study is related to Ethiopian strain and the previous Egyptian strains (Ismailia 3/Egy/2010 isolate, El-Kalubeya isolate and Ismailia 1-2014 isolate). The PPRV isolate of present study and all previous Egyptian isolates belong to lineage IV in phylogenetic analysis. The results emphasize the importance of molecular methods for a broader understanding of the epidemiology and development of the virus in the country.Key words: PPRV; Conventional RT- PCR; phylogenetic analysis; Al-Sharqia

Importance of the 3'-Terminal Nucleotide of the Forward Primer for Nucleoprotein Gene Detection of Viral Hemorrhagic Septicemia Virus by Conventional Reverse-Transcription PCR.

Viral hemorrhagic septicemia virus (VHSV), causing severe diseases in farmed fish, is detected and genotyped using conventional reverse-transcription PCR (cRT-PCR) targeting the nucleoprotein gene with corresponding VN F (forward) and VN R (reverse) primers. However, these primers have low sensitivity to VHSV subtype IVa; I investigated the cause for the poor cRT-PCR performance using various primer combinations. The results demonstrated that a 3'-end mismatch in the VN F primer reduced sensitivity and plays a critical role in VHSV detection by cRT-PCR.

A Direct Plasma miRNA Assay for Early Detection and Histological Classification of Lung Cancer

Cell-free microRNAs in plasma provide circulating biomarkers for lung cancer. Most techniques for analysis of miRNAs require a large plasma volume to purify a sufficient RNA yield followed by complicated downstream processing. Small differences in the multiple procedures often cause large analytical variations and poor diagnostic values of the plasma biomarkers. Here we investigate whether directly quantifying plasma miRNAs without RNA purification could diagnose lung cancer. FirePlex assay was directly applied to 20 μl plasma of 56 lung cancer patients and 28 cancer free controls for quantifying 11 lung tumor–associated miRNAs. FirePlex assay is easier, less expensive and time-consuming for quantification of plasma miRNAs compared with conventional reverse transcription PCR with an equivalent analytic performance. From the lung tumor–associated miRNAs, a prediction model based on two miRNAs (miRs-205-5p and -210-3p) was developed, producing 78.6% sensitivity and 89.3% specificity for identifying lung cancer. The diagnostic value was independent of stage of lung tumor, and patients’ age and sex (all P > 0.05). Furthermore, based on the same two miRNAs, additional prediction models were developed with 75.0% sensitivity and 89.3% specificity for diagnosis of lung squamous cell carcinoma, and 82.2% sensitivity and 89.3% specificity for lung adenocarcinoma. The direct plasma assay can improve the efficacy of miRNA assessment in a small plasma volume by reducing multiple procedure-associated analytical variables. The developed plasma miRNA biomarkers might be useful for the early detection and histological classification of lung cancer.

Validation of a novel one-step reverse transcription polymerase chain reaction method for detecting viral haemorrhagic septicaemia virus

Viral haemorrhagic septicaemia (VHS) is one of the most serious viral diseases in salmonid and olive flounder farms. Various diagnostic methods for detecting VHS virus (VHSV) are described in the VHS chapter of the World Organization for Animal Health (OIE) Aquatic Diagnostic Manual. A conventional reverse transcription-PCR (cRT-PCR) targeting the viral nucleocapsid gene is recommended for the detection of VHSV and, to some extent, for genotypic classification. However, the recommended assay exhibits low sensitivity for the detection of VHSV genotype IVa isolates and often shows non-specific amplicons when the RNA template is extracted from non-infected fish cell lines. For these reasons, it is necessary to develop a new RT-PCR method for the foolproof detection of all VHSV genotypes and elimination of non-specific results. In this study, we selected five candidate primer sets that target the VHSV nucleoprotein (N) gene, and selected the most sensitive among them (3F/2R). We then established the optimal reaction conditions for these primers, and ensured that no non-specific amplification had occurred in the fish tissues, fish cell lines, or heterologous viruses. The analytical sensitivity of the novel cRT-PCR was compared to that of cell culture assays, real-time RT-PCR, and other cRT-PCR methods and was found to be as sensitive as or superior to the other methods for detecting all VHSV genotypes. Our newly developed cRT-PCR assay was tested with 80 isolates, representing a collection of all known VHSV genotypes worldwide. Clear and unique amplicons were amplified from all 80 VHSV isolates. The reproducibility, and partly the robustness, of the assay were confirmed by an inter-laboratory proficiency tests including nine laboratories. A high diagnostic sensitivity and specificity was confirmed on tissue material from affected fish. In conclusion a highly robust, sensitive and specific cRT-PCR for detection of VHSV was developed and validated.