Conventional RT-PCR Method Research Articles

FASTER AND COST-EFFECTIVE EXTRACTION FREE METHOD FOR SARS-COV2 DETECTION BY RT-PCR

Background: The World Health Organization (WHO) declared an outbreak of febrile respiratory illness caused by Severe Acute Respiratory Syndrome Corona Virus-2 (SARS-CoV-2) in Wuhan City, China in December 2019. Within the immediate outbreak of three months, the global community was challenged with a devastating pandemic that caused heavy morbidities and mortalities around the globe. The expeditious spread of the virus, challenged the diagnostic laboratories to rapidly develop the accurate molecular diagnostic methods. As SARS CoV-2 assays became available for testing on existing molecular platforms, laboratories devoted unprecedented energy and resources to evaluating the analytical performance of the new tests and in some cases developed their diagnostic assays under FDA-EUA guidance. Aim: This study aims to compare the diagnostic efficacy of the extraction-free method of COVID-19 PCR and the conventional RT-PCR with extraction. Method: To get an accurate view of how an omission of RNA extraction step would perform in a real-world setting, a comparative study was performed using TATA MD CHECK RT-PCR DIRECT (Direct PCR) and LabGun Genomics for Conventional RT-PCR method. Result: From this study, direct RT-PCR correctly identified 92.3% of samples (n = 50) identified positive for SARS-CoV-2 RNA by conventional RT-PCR featuring an RNA extraction. Conclusion: Direct methods may represent a reasonable alternative to meet higher testing demands with low turnaround time as reverse transcription PCR includes traditionally time-consuming RNA extraction and purification procedures.

A novel multiplex RT-qPCR assay for simultaneous detection of bovine norovirus, torovirus, and kobuvirus in fecal samples from diarrheic calves.

Calf diarrhea results in significant economic loss and is caused by a variety of pathogens, including enteric viruses. Many of these viruses, including bovine norovirus (BNoV), bovine torovirus (BToV), and bovine kobuvirus (BKoV), are recognized as the causative agents of diarrhea; however, they remain understudied as major pathogens. We developed a multiplex reverse-transcription quantitative real-time PCR (RT-qPCR) assay for rapid and simple detection of BNoV, BToV, and BKoV. Our method had high sensitivity and specificity, with detection limits of 1 × 102 copies/μL for BNoV, BToV, and BKoV, which is a lower detection limit than conventional RT-PCR for BNoV and BKoV and identical for BToV. We tested fecal samples from 167 diarrheic calves with our multiplex RT-qPCR method. Viral detection was superior to conventional RT-PCR methods in all samples. The diagnostic sensitivity of the multiplex RT-qPCR method (100%) is higher than that of the conventional RT-PCR methods (87%). Our assay can detect BNoV, BToV, and BKoV in calf feces rapidly and with high sensitivity and specificity.

A loop-mediated isothermal amplification-enabled analytical assay for the detection of SARS-CoV-2: A review.

The number of words: 4645, the number of figures: 4, the number of tables: 1The outbreak of COVID-19 in December 2019 caused a global pandemic of acute respiratory disease, and with the increasing virulence of mutant strains and the number of confirmed cases, this has resulted in a tremendous threat to global public health. Therefore, an accurate diagnosis of COVID-19 is urgently needed for rapid control of SARS-CoV-2 transmission. As a new molecular biology technology, loop-mediated isothermal amplification (LAMP) has the advantages of convenient operation, speed, low cost and high sensitivity and specificity. In the past two years, rampant COVID-19 and the continuous variation in the virus strains have demanded higher requirements for the rapid detection of pathogens. Compared with conventional RT-PCR and real-time RT-PCR methods, genotyping RT-LAMP methodand LAMP plus peptide nucleic acid (PNA) probe detection methods have been developed to correctly identified SARS-CoV-2 variants, which is also why LAMP technology has attracted much attention. LAMP detection technology combined with lateral flow assay, microfluidic technology and other sensing technologies can effectively enhance signals by nucleic acid amplification and help to give the resulting output in a faster, more convenient and user-friendly way. At present, LAMP plays an important role in the detection of SARS-CoV-2.

A Naked-Eye Visual Reverse Transcription Loop-Mediated Isothermal Amplification with Sharp Color Changes for Potential Pen-Side Test of Foot-and-Mouth Disease Virus.

Visual loop-mediated isothermal amplification (LAMP) is qualified to be applied in the field to detect pathogens due to its simplicity, rapidity and cost saving. However, the color changes in currently reported visual reverse transcription LAMP (RT-LAMP) for foot-and-mouth disease virus (FMDV) detection are not so obvious to the naked eye, so interpretation of results is troublesome. In this study, a new naked-eye visual RT-LAMP to detect all seven distinct serotypes of FMDV was established based on the 3D genes by using pH-sensitive neutral red as the indicator, rendering a sharp contrast of color changes between the negative (light orange) and the positive (pink). Analytical sensitivity tests showed that the detection limit of the visual RT-LAMP was 104 copies/µL while those were 103 and 104 copies/µL for the RT-qPCR and conventional RT-PCR methods, respectively. Specificity tests proved that the established visual RT-LAMP assay had no cross-reactivity with other common livestock viruses. Furthermore, the analysis of 59 clinical samples showed 98.31% and 100% concordance with the RT-qPCR and the RT-PCR, respectively. The pan-serotypic FMD visual RT-LAMP assay could be suitable for a pen-side test of all seven serotypes of FMDV because the results could be easily distinguished by the naked eye without the requirement of complicated instruments and professional technicians. Hence, the novel method may have a promising prospect in field tests which exert an important role in monitoring, preventing, and controlling FMD, especially in regions with no PCR or qPCR instrument available.

Sensitive and specific clinically diagnosis of SARS-CoV-2 employing a novel biosensor based on boron nitride quantum dots/flower-like gold nanostructures signal amplification

The sudden increase of the COVID-19 outbreak and its continued growth with mutations in various forms has created a global health crisis as well as devastating social and economic effects over the past two years. In this study, a screen-printed carbon electrode reinforced with boron nitride quantum dots/flower-like gold nanostructures (BNQDs/FGNs/SPCE) and functionalized by highly specific antisense DNA oligonucleotide presents an alternative and promising solution for targeting SARS-CoV-2 RNA without nucleic acid amplification. The platform was tested on 120 SARS-CoV-2 RNA isolated from real clinical samples (60 positive and 60 negative confirmed by conventional RT-PCR method). Based on obtained quantitative results and statistical analysis (box-diagram, cutoff value, receiver operating characteristic curve, and t-test), the biosensor revealed a significant difference between the two positive and negative groups with 100% sensitivity and 100% specificity. To evaluate the quantitation capacity and detection limit of the biosensor for clinical trials, the detection performance of the biosensor for continuously diluted RNA isolated from SARS-CoV-2-confirmed patients was compared to those obtained by RT-PCR, demonstrating that the detection limit of the biosensor is lower than or comparable to that of RT-PCR. The ssDNA/BNQDs/FGNs/SPCE showed negligible cross-reactivity with RNA fragments isolated from Influenza A (IAV) clinical samples and also remained stable for up to 14 days. In conclusion, the fabricated biosensor may serve as a promising tool for point-of-care applications.

EV71 was Detected by Real-Time Fluorescence Quantitative RT-PCR Combined with Hand-Foot-Mouth Disease Rhesus Monkey Model

In this study, a real-time fluorescence quantitative RT-PCR method was developed for the detection of enterovirus 71 (EV71) and the detection of EV71 viral load during the establishment of hand-foot-mouth disease (HFMD) model in rhesus monkeys. The model of hand-foot-mouth macaque was established by injecting newborn macaques infected with the virus venom, and the dynamic changes of virus content in blood samples from 0 to 14 days after infection were monitored by real-time quantitative RT-PCR. The linear range of the standard curve was between 103 and 108 copies/µL, and the PCR amplification rate was 99.37%. The results showed that the established method could detect a minimum of 102 copies of viral RNA, 100 times higher than the conventional RT-PCR method. It has the characteristics of strong specificity, high sensitivity and good stability. The results of blood sample analysis showed that the three groups of rhesus monkey models were successfully infected with EV71, and the copy number of virus proliferation was correlated with time and challenge dose. The results showed that real-time quantitative PCR could be used for quantitative detection of EV71 viral load.

Genome characterization, prevalence and tissue distribution of astrovirus, hepevirus and norovirus among wild and laboratory rats (Rattus norvegicus) and mice (Mus musculus) in Hungary.

Rodents including rats are reservoir of several pathogens capable of affecting human health. In this study, faecal and different organ specimens from free-living Norway rats (Rattus norvegicus) (N=18) and faecal samples from laboratory rodents (rats N=21 and mice N=20) collected from different geographic areas in Hungary between 2017 and 2020 were investigated by viral metagenomics and conventional RT-PCR methods. The complete genome of three different RNA viruses, rat astrovirus, rat norovirus and rat hepevirus were characterized and analysed in detail. Rat norovirus was detected in faecal (17.6%, 3/17) and kidney (7.1%, 1/14) samples; rat astrovirus in faecal (23.5%, 4/17) and spleen (13.3%, 2/15) samples, and rat hepevirus in 43% to 67% the faecal, liver, kidney, lung, heart, muscle, brain and blood samples from Norway rats, respectively. Rat norovirus was also identifiable in 5% (1/21) of laboratory rats and rat astrovirus in 40% (8/20) of faecal samples from laboratory mice. Co-infections were found in 28% (5/18) wild Norway rats. The highest RNA viral load of astrovirus (1.81×108 copy/g) and norovirus (3.49×107 copy/g) were measured in faecal samples; while the highest RNA viral load of hepevirus (1.16×109 copy/g) was found in liver samples of Norway rats, respectively. This study confirms the wide geographic distribution and high prevalence of astrovirus, norovirus and hepevirus among wild rats in Hungary with confirmation of different organ involvement of as well as the detection of norovirus and astrovirus in laboratory rats and mice, respectively. This finding further strengthens the role of rodents in the spread of viral pathogens especially infecting human.

Reverse transcriptase loop-mediated isothermal amplification (RT-LAMP)-based diagnosis: A potential alternative to quantitative real-time PCR based detection of the novel SARS-COV-2 virus

The sudden outbreak of the novel Coronavirus infectious disease (COVID-19) resulted in significant challenges to global health systems. One of the primary challenges is rapid, reliable, and accurate detection of the severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) virus among the suspected COVID-19-infected individuals. At present, quantitative real-time PCR (qRT-PCR) is a widely used diagnostic method. However, it requires expensive instruments and expertise in the interpretation of results. These constraints reflect the significant need for the development of alternative diagnostic options.This study will validate the use and efficiency of the reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay as a potential alternative for the detection of COVID-19. For this purpose, a cohort of 297 suspected COVID-19 patients was tested using both the RT-LAMP assay and the conventional RT-PCR method. For the RT-LAMP assay, three genes (orf-1ab, N, and S) were identified as the target sites for the detection of COVID-19. Based on a comparative assessment, 117 out of 124 positive COVID-19 cases were observed using the RT-LAMP technique with an overall 91.45% sensitivity. Interestingly, where a consensus on 163 individuals free of SARS-Cov-2 was observed, RT-LAMP specificity was 90%. Based on these findings, the robustness of the technique, and the reduced dependency on expensive instrumentation, RT-LAMP-based COVID-19 detection is strongly recommended as a potential alternative assay.

Genetic diversity of tilapia lake virus genome segment 1 from 2011 to 2019 and a newly validated semi-nested RT-PCR method

The gene of RNA viruses, encoding RNA-directed RNA polymerase (RdRp) is relatively conserved due to its crucial function in viral genome replication and transcription making it a useful target for genetic diversity study and PCR detection. In this study, we investigated the genetic diversity of 21 tilapia lake virus (TiLV) genome segment 1 sequences predictively coding for RdRp subunit P1. Those sequences were obtained from infected fish samples collected in Ecuador, Israel, Peru, and Thailand between 2011 and 2019 (nine sequences from this study and 12 sequences from GenBank). Primers were then designed from the highly conserved regions among all 21 TiLV segment 1 sequences and used in semi-nested RT-PCR condition optimization. The result revealed that all 21 TiLV segment 1 sequences showed 95.00–99.94 and 99.00–100% nucleotide and amino acid sequence identity, respectively. These isolates were phylogenically clustered into three separate genetic clades, called i) Israeli-2011 clade (containing of TiLV isolates from Israel collected in 2011, Ecuador, and Peru isolates), ii) Israeli-2012 clade (containing only TiLV isolates collected from Israel in 2012), and iii) Thai clade (containing only sequences obtained from Thailand isolates). The newly established PCR protocol was 100 times more sensitive than our previous segment 3-based protocol when comparatively assayed with RNA extracted from infected fish. The assay was also shown to be specific when tested against negative control samples, i.e. RNA extracted from clinical healthy tilapia and from bacterial and viral pathogens (other than TiLV) commonly found in aquatic animals. Validation experiment with RNA extracted from naturally infected fish specimens collected in 2013–2019 yielded positive test results for all samples tested, confirming that the newly designed primers and detection protocol against TiLV segment 1, is an alternative option for TiLV diagnosis in laboratories where conventional RT-PCR method is preferred.

Highly Sensitive Gold Nanoparticle Polymerase Chain Reaction in the Detection of Anaplastic Lymphoma Kinase-Positive Gastric Cancer from a Biopsy Specimen

Gastric cancer is a malignant tumor that is originated from the epithelia of the gastric mucosa. Although the gastroscopic biopsy of suspicious gastric areas can provide better early-warning opportunities for patients with malignant tumors that result in achieving early diagnosis and treatment, many malignant tumors are still excluded due to atypical histology, sampling errors, nonspecific antibodies, and other reasons, which pose a threat to the physical and mental health of patients. Therefore, more sensitive and specific detection methods of gastric cancer are needed to improve the screening efficiency of gastroscopic biopsy. The sensitivity of nano PCR was 10 times higher than that of conventional PCR. In comparison with the conventional RT-PCR method, nano PCR technology can amplify brighter bands and identify higher gene expression levels for ALK weak positive gastric cancer in IHC, and improve the detection rate of clinical specimens with lower ALK staining. Therefore, nano-gold polymerase chain reaction used gold nanoparticle (nano-gold PCR) has high sensitivity and positive detection rate for ALK-positive gastric cancer identified by gastroscopy. When a low concentration was observed for the amplified gene, especially when the biopsy tissue was too small to carry out IHC staining, the target gene could be amplified more effectively. Therefore, nano PCR technology is proposed to be widely used in target gene detection of biopsy tissue to achieve better tumor screening.

Dual priming oligonucleotide (DPO)-based real-time RT-PCR assay for accurate differentiation of four major viruses causing porcine viral diarrhea

Currently in China, porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine rotavirus (PoRV), and porcine deltacoronavirus (PDCoV) are the major causes of porcine viral diarrhea, and mixed infections in clinics are common, resulting in significant economic losses in pig industry. Here, a dual priming oligonucleotide (DPO)-based multiplex real-time SYBR Green RT-PCR assay were developed for accurately differentiating PEDV, TGEV, PoRV, and PDCoV in clinical specimens targeting the N gene of TGEV, PEDV, and PDCoV, and the VP7 gene of PoRV. Results showed that the DPO primer allowed a wider annealing temperature range (40–65 °C) and had a higher priming specificity compared to conventional primer, in which more than 3 nucleotides in the 3′- or 5′-segment of DPO primer mismatched with DNA template, PCR amplification efficiency would decrease substantially or extension would not proceed. DPO-based multiplex real-time RT-PCR method had analytical detection limit of 8.63 × 102 copies/μL, 1.92 × 102 copies/μL, 1.74 × 102 copies/μL, and 1.76 × 102 copies/μL for PEDV, TGEV, PoRV, and PDCoV in clinical specimens, respectively. A total of 672 clinical specimens of piglets with diarrheal symptoms were collected in Northeastern China from 2017 to 2018 followed by analysis using the assay, and epidemiological investigation results showed that PEDV, TGEV, PoRV, and PDCoV prevalence was 19.05%, 5.21%, 4.32%, and 3.87%, respectively. The assay developed in this study showed higher detection accuracy than conventional RT-PCR method, suggesting a useful tool for the accurate differentiation of the four major viruses causing porcine viral diarrhea in practice.

A multiplex xTAG assay for the simultaneous detection of five chicken immunosuppressive viruses

BackgroundChicken anemia virus (CAV), avian reovirus (ARV), infectious bursal disease virus (IBDV), Marek’s disease virus (MDV) and reticuloendotheliosis virus (REV) all cause immunosuppressive disease in birds through vertical or horizontal transmission. Mixed infections with these immunosuppressive pathogens lead to atypical clinical signs and obstruct accurate diagnoses and epidemiological investigations. Therefore, it is essential to develop a high-throughput assay for the simultaneous detection of these immunosuppressive viruses with high specificity and sensitivity. The aim of this study was to establish a novel method using a RT-PCR assay combined with fluorescence labeled polystyrene bead microarray (multiplex xTAG assay) to detect single or mixed viral infections.ResultsThe results showed that the established xTAG assay had no nonspecific reactions with avian influenza virus (AIV), infectious bronchitis virus (IBV), newcastle disease virus (NDV), infectious laryngotracheitis virus (ILTV), Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS). The limit of detection was 1.0 × 103 copies/μL for IBDV and 1.0 × 102copies/μL for the other four viruses. Ninety field samples were tested and the results were confirmed using conventional RT-PCR methods. The detection results of these two methods were 100% consistent. The established multiplex xTAG assay allows a high throughput and simultaneous detection of five chicken immunosuppressive viruses.ConclusionThe multiplex xTAG assay has been showed to be an additional tool for molecular epidemiology studies of five chicken immunosuppressive viruses in the poultry industry.

Rapid detection of Banna virus by reverse transcription-loop-mediated isothermal amplification (RT-LAMP)

ObjectivesBanna virus (BAV) is classified in the genus Seadornavirus within the Reoviridae family and considered to be an emerging pathogen. We aimed to develop a rapid and simple molecular detection approach for all BAV subgroups in isothermal conditions. MethodA set of six specific primers was designed to target the segment 12 of BAV, and the reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay was developed and compared with conventional RT-PCR method. ResultsThe amplification of the RT-LAMP assay can be obtained within 40min at 65°C. The results from specificity showed that only target BAVs RNA including genotypes A, B and C were amplified and the assay demonstrated a sensitivity of 3.6×10−2PFU/mL, which was higher than conventional RT-PCR measurement. A good reliability for the assay was presented in the further evaluation for BAVs RNA from serial diluted BAV-spiked serum and 47 pools of field mosquito samples. ConclusionsOur findings present a rapid, sensitive and specific RT-LAMP assay that can be applied for BAV detection in clinical or field samples in the future.

EvaGreen-based real-time PCR assay for sensitive detection of salmonid alphavirus

Salmonid alphaviruses (SAVs), which include the etiological agents of salmon pancreas disease (PD) and sleeping disease (SD), are significant viral pathogens of European salmonid aquaculture, resulting in substantial economic losses to the salmonid-farming industry. Even though many countries including China have not reported the presence of SAV infections, these countries may be seriously threatened by these diseases as the salmon fish import trade increases. Thus, it is indeed necessary to develop efficient detection methods for the diagnosis and prevention of SAV infection. Real-time PCR assays have been increasingly used in viral detection, and in many cases scientists prefer dye-based real-time PCR assays for their high sensitivity and low cost. In this study, we developed a novel, sensitive, low-cost detection method, EvaGreen-based real-time PCR assay for the detection of SAV. This assay exhibited high specificity for SAV1, SAV2, and SAV5 and was able to detect SAV at concentrations as low as 1.5 × 101 copies, making them more sensitive than the approved conventional RT-PCR method (detection limit, 1.5 × 106 copies). Assessment of infected fish samples showed that the sensitivity of EvaGreen-based assay was higher than previously developed SYBR Green assay (227 assay). Thus, we report that the EvaGreen real-time PCR assays is an economical alternative diagnostic method for the rapid detection of SAV1, SAV2, and SAV5 infection, providing improved technical support for the clinical diagnosis and epidemiological investigation of SAV.

Detection of 22 common leukemic fusion genes using a single-step multiplex qRT-PCR-based assay

BackgroundFusion genes generated from chromosomal translocation play an important role in hematological malignancies. Detection of fusion genes currently employ use of either conventional RT-PCR methods or fluorescent in situ hybridization (FISH), where both methods involve tedious methodologies and require prior characterization of chromosomal translocation events as determined by cytogenetic analysis. In this study, we describe a real-time quantitative reverse transcription PCR (qRT-PCR)-based multi-fusion gene screening method with the capacity to detect 22 fusion genes commonly found in leukemia. This method does not require pre-characterization of gene translocation events, thereby facilitating immediate diagnosis and therapeutic management.MethodsWe performed fluorescent qRT-PCR (F-qRT-PCR) using a commercially-available multi-fusion gene detection kit on a patient cohort of 345 individuals comprising 108 cases diagnosed with acute myeloid leukemia (AML) for initial evaluation; remaining patients within the cohort were assayed for confirmatory diagnosis. Results obtained by F-qRT-PCR were compared alongside patient analysis by cytogenetic characterization.ResultsGene translocations detected by F-qRT-PCR in AML cases were diagnosed in 69.4% of the patient cohort, which was comparatively similar to 68.5% as diagnosed by cytogenetic analysis, thereby demonstrating 99.1% concordance. Overall gene fusion was detected in 53.7% of the overall patient population by F-qRT-PCR, 52.9% by cytogenetic prediction in leukemia, and 9.1% in non-leukemia patients by both methods. The overall concordance rate was calculated to be 99.0%. Fusion genes were detected by F-qRT-PCR in 97.3% of patients with CML, followed by 69.4% with AML, 33.3% with acute lymphoblastic leukemia (ALL), 9.1% with myelodysplastic syndromes (MDS), and 0% with chronic lymphocytic leukemia (CLL).ConclusionsWe describe the use of a F-qRT-PCR-based multi-fusion gene screening method as an efficient one-step diagnostic procedure as an effective alternative to lengthy conventional diagnostic procedures requiring both cytogenetic analysis followed by targeted quantitative reverse transcription (qRT-PCR) methods, thus allowing timely patient management.

Rapid and sensitive detection of salmonid alphavirus using TaqMan real-time PCR

Salmonid alphavirus (SAV) infection has led to the spread of salmon pancreas disease (PD) and sleeping disease (SD) to salmonids in several countries in Europe, resulting in tremendous economic losses to the fish farming industry. Recently, with increases in the fish import trade, many countries in which SAV has been unreported, such as China, may be seriously threatened by these diseases. It is therefore necessary to develop efficient detection methods for the prevention and diagnosis of SAV infection. In this study, a rapid and sensitive TaqMan real-time PCR method was established and assessed for this purpose. A specificity assay showed no cross-reactions with other common RNA viruses. Regression analysis and standard curves calculated from the Ct values of 10-fold serial dilutions of the standard plasmid showed that the assay was highly reproducible over a wide range of RNA input concentrations. The real-time PCR assay was able to detect SAV at a concentration as low as 1.5 × 101 copies, indicating that it is 107 times more sensitive than the approved conventional RT-PCR method (detection limit, 1.5 × 107 copies) after use on the same samples. Assessment of infected fish samples showed that this assay has a higher sensitivity than the previously reported Q_nsP1 assay. Thus, this TaqMan real-time PCR assay provides a rapid, sensitive, and specific detection method for SAV, offering improved technical support for the clinical diagnosis and epidemiology of SAV.

Effects of Plasma Exchange (PE) Therapy in a Severe Fever with Thrombocytopenia Syndrome (SFTS) Patient with High Virus Quantities

Background: Severe fever with thrombocytopenia syndrome (SFTS) is an infectious disease caused by the SFTS bunyavirus, which is carried by ticks, and presents various symptoms such as fever, fatigue, digestive symptoms, and hemophagocytic lymphhistiocytosis (HLH). Since the first case was reported from China in 2011, there has been a rapid increase of newly diagnosed patients in Japan. Because the mortality rate has remained high (10-30%), there is an urgent need to establish a management strategy for SFTS.Method: We retrospectively reviewed two newly-diagnosed cases of severe SFTS in our hospital from April 2015 to July 2015. Diagnosis of SFTS was confirmed rapidly using a conventional one-step RT-PCR (cvPCR) method, and the quantity of SFTS virus in each serum was determined with a quantitative one-step RT-PCR (qPCR) method described by Yoshikawa et al. (JCM2014, 52, 3325-33). Virus titer is indicated in units of log10 copies/ml.Results: Case 1 was an 87-year-old female and Case 2 was an 81-year-old male. They showed similar severe clinical symptoms including disturbance of consciousness and a variety of abnormal laboratory findings. Both cases were quickly confirmed as SFTS using cvPCR on the next day after blood sampling. Bone marrow (BM) aspiration was performed at diagnosis, and confirmed HLH in both cases.[Case 1]Although methylprednisolone (mPSL) 250 mg was administrated to control HLH on day 3, the data indicated that HLH had become worse on day 4. To control HLH, mPSL pulse therapy (1000 mg/day) was administrated from day four to day six. However, multiple organ failure proceeded rapidly, and she expired on day eight. Serum virus quantities increased markedly from 6.99 (day 3) to 9.05 (day 6). In this case, the virus level at diagnosis was extremely high compared to previously reported cases and it was possible that the high-dose mPSL therapy suppressed anti-virus immunity.[Case 2]Although mPSL 500 mg was administrated from day two, organ damage progressed on day three. In this case, instead of increasing the dosage of mPSL, plasma exchange (PE) was performed from day four to day six, and organ damage was relieved and the patient recovered in good condition. The serum virus quantities decreased slowly from 6.62 (day 4) to 6.48 (day 7) after PE.Discussion: Yoshikawa et al. reported that virus level at diagnosis correlated to the outcomes of SFTS patients and that in most fatal cases virus quantities at diagnosis were over 5.0. In our cases, the patients had higher virus quantities at diagnosis and presented with severe symptoms. In the first case of fatality, the virus quantities remarkably increased shortly after mPSL therapy. As most SFTS patients are over the age of 50 and the mortality rate is higher in the elderly, lowered immunity in elderly patients might be the cause of SFTS progression. Moreover, immunosuppression therapy, including high dose mPSL therapy for HLH, might enhance the accelerated proliferation of SFTS virus. Although mPSL therapy is effective in some cases, immunosuppressive therapy for a SFTS patient is still controversial. In the second case, after PE therapy the virus level didn't increase and symptoms were improved. PE therapy does not suppress humoral and cellular immunity, which is different from mPSL therapy. As such, PE therapy might be effective in serious cases of SFTS with high virus quantities. In these cases, treatment plans were developed without information on virus quantities, and we obtained qPCR results afterwards. In the future, if qPCR can be performed as quickly as cvPCR, then data on the virus level at diagnosis may be the most important information for planning treatment. DisclosuresNo relevant conflicts of interest to declare.

Diagnosis of Cetacean morbillivirus: A sensitive one step real time RT fast-PCR method based on SYBR® Green

Cetacean morbillivirus (CeMV) (family Paramyxoviridae, genus Morbillivirus) is considered the most pathogenic virus of cetaceans. It was first implicated in the bottlenose dolphin (Tursiops truncatus) mass stranding episode along the Northwestern Atlantic coast in the late 1980s, and in several more recent worldwide epizootics in different Odontoceti species. This study describes a new one step real-time reverse transcription fast polymerase chain reaction (real-time RT-fast PCR) method based on SYBR® Green to detect a fragment of the CeMV fusion protein gene. This primer set also works for conventional RT-PCR diagnosis. This method detected and identified all three well-characterized strains of CeMV: porpoise morbillivirus (PMV), dolphin morbillivirus (DMV) and pilot whale morbillivirus (PWMV). Relative sensitivity was measured by comparing the results obtained from 10-fold dilution series of PMV and DMV positive controls and a PWMV field sample, to those obtained by the previously described conventional phosphoprotein gene based RT-PCR method. Both the conventional and real-time RT-PCR methods involving the fusion protein gene were 100- to 1000-fold more sensitive than the previously described conventional RT-PCR method.

DNA 교차 오염 방지 기능을 가진 돼지 인플루엔자바이러스 감별진단용 one-step multiplex RT-PCR 진단법

Abstract In this study, we developed a cost and time saving one-step multiplex RT-PCR for the simultaneous detection and differentiation of swine influenza viruses (SIV) and 2009 pandemic influenza H1N1 virus (pH1N1). The one-step multiplex RT-PCR using four sets of primer was confirmed to be capable of detection of all SIV subtypes and differential diagnosis of major SIV subtype H1, H3 and pH1N1 on individual or mixed viral culture samples. The sensitivity of the multiplex RT-PCR was determined to be at least 2 −6 HA/25 L of the presented SIVs, providing sufficient efficacy for a routine SIV monitor-ing in diagnostic laboratories. In addition, compared with the conventional RT-PCR methods that cannot avoid the carryover DNA contamination, the developed RT-PCR applied with the uracil DNA glyco-sylase (UNG) system was proven to prevent a false positive reaction by carryover contamination of the pre-amplified DNA. In conclusion, the one-step RT-PCR with UNG system could be applicable to de-tect and differentiate of SIV from the viral cultures without worry of carryover DNA contamination in clinical laboratories.

Development of PCR methods for detection of EAV infection

The goal of this work was the development of suitable (real-time) RT-PCR techniques for fast and sensitive diagnosis of EAV and for molecular-epidemiological characterisation of viral strains, as an alternative to virus isolation. To this purpose two conventional RT-PCR methods and one real-time RT-PCR were adapted to detect the broadest possible spectrum of viral strains. Several dilutions with Bucyrus strain showed a 100-fold higher sensitivity of real-time RT-PCR and heminested RT-PCR compared to simple RT-PCR. Making use of 11 cell culture supernatants of different EAV isolates and 7 semen samples of positive stallions, the suitability of the techniques could be shown. Phylogenetic analysis of sequences of the newly analysed samples compared with known sequences indicated that more EAV-lineages exist than presently described.