• Food Science of Animal Resources
• /
• 제31권6호
• /
• pp.851-857
• /
• 2011

This study was conducted to develop a real time reverse transcriptase-PCR (RT-PCR) method for the detection of viable Enterobacteriaceae in milk using primers based on the genes of ribosomal proteins S11 and S13 and to determine effects of heating and subsequent treatments on the threshold cycle (Ct) of the real time RT-PCR. Total RNA was isolated from 17 strains of bacteria including 11 strains of Enterobacteriaceae suspended in milk using a modified Tri reagent method. SYBR Green Master Mix was added to the RNA and the mixture was subjected to the real time RT-PCR. The Cts of eleven type strains of the Enterobacteriaceae in milk ($10^7$ cells) in the real time RT-PCR ranged from 21.5 to 24.6. However, the Cts of Pseudomonas fluorescens, Acinetobacter calcoaceticus, and three gram-positive bacteria were more than 40. The real time RT-PCR detected as low as $10^3$ cells in agarose gel electrophoresis. The Cts increased from 22.0 to 34.2 when milk samples contaminated with Escherichia coli ($10^7$ cells/mL) were heated at $65^{\circ}C$ for 30 min. In addition, subsequent incubation at $37^{\circ}C$ for 6 and 24 h increased the Cts further up to 36.2 and 37.2, respectively. Addition of RNase A to the bacterial suspension obtained from the heated milk and subsequent incubation at $37^{\circ}C$ for 1 h increased the Cts to more than 40. The results of this study suggests that pretreatment of bacterial cells heated in milk with RNase A before RNA extraction might enhance the ability to differentiate between viable and dead bacteria using real time RT-PCR.

• Journal of Veterinary Clinics
• /
• 제23권3호
• /
• pp.300-307
• /
• 2006

In the present study, methods of the reverse transcription-polymerase chain reaction(RT-PCR) were evaluated for the rapid detection and differentiation of transmissible gastroenteritis virus(TGEV), porcine epidemic diarrhea virus(PEDV) and rotavirus in piglets suffering from diarrhea. For the purposes, the PCR conditions were first confirmed for the amplification of VP7 gene of rotavirus and N gene of TGEV and PEDV using each specific primers and their annealing temperature. Multiplex RT-PCR methods were further determined to distinguish these viral infections and the results are as follows. For the specific amplification of these viral genes, the reliable PCR condition was determined as 30 cycles of reaction consisting each 1 min of denature at $94^{\circ}C$, annealing at $42^{\circ}C$ and polymerization at $72^{\circ}C$ with 1.0 mM $MgCl_2$. It was able to differentiate these viral infections in the intestines and feces of piglets suffering from diarrhea by duplex PCR for TGEV and PEDV and single PCR for rotavirus with a primer-annealing temperature of $42^{\circ}C$. When the multiplex RT-PCR were undertaken for the field samples, 17 cases of PEDV and 5 cases of rotavirus infections were differential diagnosed in a total of 92 samples of intestines and feces of the piglets with diarrhea.

• Microbiology and Biotechnology Letters
• /
• 제43권3호
• /
• pp.267-274
• /
• 2015

Biopharmaceuticals and the cell substrates used for their manufacture are currently tested for porcine adventitious viruses due to the widespread use of porcine trypsin in cell culture. Porcine transmissible gastroenteritis virus (PTGV) is one of the major adventitious porcine viruses causing contaminated during the manufacture of biopharmaceuticals. Therefore, rapid and sensitive detection of PTGV is essential in ensuring the safety of biopharmaceuticals. A TaqMan probe real-time RT-PCR method was developed for the quantitative detection of PTGV contamination in cell substrates, raw materials, manufacturing processes, and final products, as well as PTGV clearance validation. Specific primers for the amplification of PTGV RNA were selected, and PTGV RNA was quantified by use of a specific TaqMan probe. Specificity, limit of detection (LOD), and robustness of the method was validated according to international guidelines on the validation of nucleic acid amplification tests. The sensitivity of the assay was calculated to be 1.10 × 10⁰ TCID₅₀/ml. The real-time RT-PCR method was validated to be reproducible, very specific to PTGV, and robust. The established real-time RT-PCR assay was successfully applied to the validation of Chinese Hamster Ovary (CHO)-K1 cells artificially infected with PTGV.

• Korean Journal of Veterinary Service
• /
• 제37권4호
• /
• pp.263-271
• /
• 2014

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 $2^{-6}$ $HA/25{\mu}L$ of the presented SIVs, providing sufficient efficacy for a routine SIV monitoring 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 glycosylase (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 detect and differentiate of SIV from the viral cultures without worry of carryover DNA contamination in clinical laboratories.

• Korean Journal of Microbiology
• /
• 제49권3호
• /
• pp.270-274
• /
• 2013

The bumblebee, Bombus terrestris, has played an important role as one of the alternative pollinators since the outbreak of honeybee collapse disorder. Recently, pathogens and parasites such as viruses, bacteria and mites, which affect the life span and fecundity of their host, have been discovered in B. terristris. In order to detect the microsporidian pathogen, Nosema spp. in the field populations of B. terristris, we collected adults and isolated their genomic DNA for diagnostic PCR. The PCR primers specific for Nosema spp. were newly designed and applied to gene amplification for cloning. Only small subunit ribosomal RNA (SSU rRNA) gene of N. ceranae was successfully amplified among examined genes and sequenced, which indicates that N. ceranae mainly infects the examined field population of B. terristris. To detect of SSU rRNA gene, two regions of SSU rRNA gene were selected by primary PCR analysis and further analyzed in quantitative real-time PCR (qRT-PCR). The qRT-PCR analysis demonstrated that SSU rRNA of N. ceranae was detected at concentration as low as $0.85ng/{\mu}l$ genomic DNA. This result suggests that the detection via qRT-PCR can be applied for the rapid and sensitive diagnosis of N. ceranae infection in the field population as well as risk assessment of B. terristris.

• Korean Journal of Radiology
• /
• 제21권10호
• /
• pp.1150-1160
• /
• 2020

Objective: To describe the experience of implementing a deep learning-based computer-aided detection (CAD) system for the interpretation of chest X-ray radiographs (CXR) of suspected coronavirus disease (COVID-19) patients and investigate the diagnostic performance of CXR interpretation with CAD assistance. Materials and Methods: In this single-center retrospective study, initial CXR of patients with suspected or confirmed COVID-19 were investigated. A commercialized deep learning-based CAD system that can identify various abnormalities on CXR was implemented for the interpretation of CXR in daily practice. The diagnostic performance of radiologists with CAD assistance were evaluated based on two different reference standards: 1) real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) results for COVID-19 and 2) pulmonary abnormality suggesting pneumonia on chest CT. The turnaround times (TATs) of radiology reports for CXR and rRT-PCR results were also evaluated. Results: Among 332 patients (male:female, 173:159; mean age, 57 years) with available rRT-PCR results, 16 patients (4.8%) were diagnosed with COVID-19. Using CXR, radiologists with CAD assistance identified rRT-PCR positive COVID-19 patients with sensitivity and specificity of 68.8% and 66.7%, respectively. Among 119 patients (male:female, 75:44; mean age, 69 years) with available chest CTs, radiologists assisted by CAD reported pneumonia on CXR with a sensitivity of 81.5% and a specificity of 72.3%. The TATs of CXR reports were significantly shorter than those of rRT-PCR results (median 51 vs. 507 minutes; p < 0.001). Conclusion: Radiologists with CAD assistance could identify patients with rRT-PCR-positive COVID-19 or pneumonia on CXR with a reasonably acceptable performance. In patients suspected with COVID-19, CXR had much faster TATs than rRT-PCRs.

• Korean Journal of Food Science and Technology
• /
• 제37권6호
• /
• pp.1018-1023
• /
• 2005

Simplified procedure was developed for concentrating and detecting poliovirus and norovirus in oysters. Viruses were seeded into oyster tissue homogenates and concentrated through polyethylene glycol (PEG) precipitation, chloroform or Freon extraction, with additional PEG precipitation. Amount of viruses was evaluated using poliovirus plaque assay. Virus recovery during concentration procedure was approximately 16.4-26.0%. For defection, viral RNAs in oysters were examined using one-step RT-PCR after extraction with Trizol. Dilution or capturing of viral RNA using silica gel membrane allowed viruses to be detected by RT-PCR, whereas viruses could not be removed using $QIAshredder^{TM}$ Homogenizer, which is effective in removing RT-PCR inhibitors in lettuce and hamburgers. Freon extraction, generally used to concentrate viruses found in food, could be substituted with chloroform extraction using this procedure; no difference could be observed between detection limits of whole oyster extracts and digestive organ extracts indicating that RT-PCR inhibitors were distributed evenly throughout whole tissues. Nested PCR greatly improved efficiency of this procedure. Overall, this procedure could remove sufficient amount of inhibitors to allow detection of norovirus in oysters.

• Pediatric Infection and Vaccine
• /
• 제25권2호
• /
• pp.91-100
• /
• 2018

Purpose: This study investigated the factors affecting the use of empirical antibiotics in febrile infants from 1 month to less than 3 months. Methods: We retrospectively reviewed the medical records of hospitalized previously healthy infants with fever in Pusan National University Children's Hospital from January 2010 to December 2016. Clinical features, laboratory findings and antibiotic therapy were analyzed. Respiratory viruses were identified by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) and were reported after 1-3 days. Enterovirus were identified by real time polymerase chain reaction (PCR) and were reported in several hours. Results: The 129 of 366 subjects used empirical antibiotics and 237 patients didn't used empirical antibiotics. Empirical antibiotics were used more frequently when the fever was longer before admission, respiratory symptoms and ill being appearances were present and C-reactive protein was elevated. The rate of readmission was low in the group not used empirical antibiotics. Most of the patients detected by enterovirus PCR in cerebrospinal fluid didn't used empirical antibiotics. The results of respiratory virus multiplex RT-PCR showed no difference in the use of empirical antibiotics. Conclusions: In our study, empirical antibiotic prescriptions were affected not respiratory virus multiplex RT-PCR but enterovirus PCR. If multiplex RT-PCR were reported more rapid turn around time, it will affect antibiotic use.

• Research in Plant Disease
• /
• 제7권2호
• /
• pp.80-85
• /
• 2001

This study was carried out to examine tne incidences of virus diseases in lily plants cultivated in highland areas, and to develop an effective detection method. Viral symptoms on lilies in the highland areas were differentiated into mosaic, crinkle, mottle, stripe and line pattern. The distribution of symptoms on infected plants was 43.8% of mosaic, 29.2% of crinkle, and 10.9% of mottle symptoms. Six viruses such as Lily symptomless vires(LSV), Cucumber mosaic virus (CMV), Lily mottle virus (LMoV), Lily virus X (LVX, Potexvirus), Tabacco mosaic virus (TMV,Tobamovirus), and Tabacco rattle virus (TRV,Tobravirus) were detected from the infected lilies. Infection rate of Lilium oriental (cvs. Casablanca and Marcopolo) was 2~4 times higher than that of L. asiatic (cvs. Solemio and Prato). Virus detection on lilies by one-step RT-PCR (by using reverse transcription and polymerase chain reaction simultaneously) was more rapid rapid and reliable than by the conventional RT-PCR method.

• Journal of Microbiology and Biotechnology
• /
• 제31권12호
• /
• pp.1709-1715
• /
• 2021

Outbreaks of food poisoning due to the consumption of norovirus-contaminated shellfish continue to occur. Male-specific (F+) coliphage has been suggested as an indicator of viral species due to the association with animal and human wastes. Here, we compared two methods, the double agar overlay and the quantitative real-time PCR (RT-PCR)-based method, for evaluating the applicability of F+ coliphage-based detection technique in microbial contamination tracking of shellfish samples. The RT-PCR-based method showed 1.6-39 times higher coliphage PFU values from spiked shellfish samples, in relation to the double agar overlay method. These differences indicated that the RT-PCR-based technique can detect both intact viruses and non-particle-protected viral DNA/RNA, suggesting that the RT-PCR based method could be a more efficient tool for tracking microbial contamination in shellfish. However, the virome information on F+ coliphage-contaminated oyster samples revealed that the high specificity of the RT-PCR- based method has a limitation in microbial contamination tracking due to the genomic diversity of F+ coliphages. Further research on the development of appropriate primer sets for microbial contamination tracking is therefore necessary. This study provides preliminary insight that should be examined in the search for suitable microbial contamination tracking methods to control the sanitation of shellfish and related seawater.