• Asian-Australasian Journal of Animal Sciences
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• v.23 no.10
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• pp.1291-1298
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• 2010

The aim of this research was to investigate the dynamic changes of the level of total prolactin receptor (PRLR) mRNA and the short form prolactin receptor (S-PRLR) mRNA in skin of cashmere goats from the initiation of cashmere fibre growth to active growth. Eighteen half-sib wethers were allocated randomly to two groups. Melatonin implants were used in order to initiate growth of cashmere fibre before the normal time and reduce blood plasma prolactin (PRL) concentration. Real-time reverse transcription quantitative polymerase chain reaction (real-time PCR) was used to determine PRLR mRNA expression levels of skin from June to November. The results showed that, in Chinese Inner Mongolia cashmere goats, there were seasonal variations in expression of total PRLR mRNA in skin with levels decreasing from June to October. Synchronously, the cashmere fibre growth rate gradually increased during this period, but the expression levels of S-PRLR mRNA did not decrease along with seasonal variation from initiation to active growth of cashmere fibre. These results suggest that expression levels of S- PRLR mRNA might be involved in the process of cashmere growth. It was also possible that the change of alternative splicing of PRLR occurred in the skin of cashmere goats from proanagen to anagen.

• The Plant Pathology Journal
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• v.39 no.4
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• pp.309-318
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• 2023

Huanglongbing (HLB) is one of the most destructive diseases in citrus, which imperils the sustainability of citriculture worldwide. The presumed causal agent of HLB, 'Candidatus Liberibacter asiaticus' (CLas) is a non-culturable phloem-limited α-proteobacterium transmitted by Asian citrus psyllids (ACP, Diaphorina citri Kuwayama). A widely adopted method for HLB diagnosis is based on quantitative real-time polymerase chain reaction (qPCR). Although HLB diagnostic qPCR provides high sensitivity and good reproducibility, it is limited by time-consuming DNA preparation from plant tissue or ACP and the requirement of proper lab instruments including a thermal cycler to conduct qPCR. In an attempt to develop a quick assay that can be deployed in the field for CLas detection, we developed a real-time loop-mediated isothermal amplification (rt-LAMP) assay by targeting the CLas five copy nrdB gene. The rt-LAMP assay using various plant sample types and psyllids successfully detected the nrdB target as low as ~2.6 Log10 copies. Although the rt-LAMP assay was less sensitive than laboratory-based qPCR (detection limit ~10 copies), the data obtained with citrus leaf and bark and ACP showed that the rt-LAMP assay has >96% CLas detection rate, compared to that of laboratory-based qPCR. However, the CLas detection rate in fibrous roots was significantly decreased compared to qPCR due to low CLas titer in some root DNA sample. We also demonstrated that the rt-LAMP assay can be used with a crude leaf DNA extract which is fully deployable in the field for quick and reliable HLB screening.

• Biomedical Science Letters
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• v.17 no.3
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• pp.191-196
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• 2011

Noroviruses (noroV) are the major cause of nonbacterial gastroenteritis in humans worldwide. Since noroV cannot yet be cultured in vitro and their diagnosis by electron microscopy requires at least $10^6$ viral particles/g of stool a variety of molecular detection techniques represent an important step towards the detection of noroV. In the present study, we have applied real-time nucleic acid sequence-based amplification (real-time NASBA) for simultaneous detection of NoroV genogroup I (GI) and genogroup II (GII) using standard viral RNA. For real-time NASBA assay which can detected noroV GI and GII, a selective region of the genes encoding the capsid protein was used to design primers and genotype-specific molecular beacon probes. The specificity of the real-time NASBA using newly designed primers and probes were confirmed using standard viral RNA of noroV GI and GII. To determine the sensitivity of this assay, serial 10-fold dilutions of standard viral RNA of noroV GI and GII were used for reverse transcription polymerase chain reaction (RT-PCR) and real-time NASBA. The results showed that while agarose gel electrophoresis could detect RT-PCR products with 10 pg of standard viral RNA, the real-time NASBA assay could detect 100 fg of standard viral RNA. These results suggested that the real-time NASBA assay has much higher sensitivity than conventional RT-PCR assay. This assay was expected that might detect the viral RNA in the specimens which could have been false negative by RT-PCR. There were needed to perform real-time NASBA with clinical specimens for evaluating accurate sensitivity and specificity of this assay.

• Journal of Life Science
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• v.20 no.8
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• pp.1201-1206
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• 2010

We performed a comparative analysis using a Real-time PCR (Polymerase Chain Reaction) and LC/MS (Liquid-Chromatograph/Mass Spectrometer) method in order to detect microcystin in environmental sources. Among the three different primer sets tested for microcystin using three positive strains of Microcystis aeruginosa by Real-time PCR assay, only TOX2P/TOX2M primer pairs were able to detect Microcystis aeruginosa. According to the results of a survey carried out from June 2009 to September 2009, 11 out of 11 (100%) raw water samples were were found to have microcystin when the Real-Time PCR and LC/MS method was used, with total microcystin concentration ranging from 5.98~219.0 ${\mu}g/l$. A microcystin removal treatment process was used to ensure entire removal, by passing it through a BAC filtration step. It was concluded that real-time PCR assay can be used to estimate micrucystin detection more rapidly and easily than the LC/MS method.

• Journal of Dairy Science and Biotechnology
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• v.33 no.4
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• pp.253-262
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• 2015

The authentication and implications of misleading labeling in milk and dairy products is important to protect against cheating consumers from adulteration and to alert sensitive consumers to any undeclared potential allergens. This need to support milk and dairy products labeling has led to the development of specific analytical techniques for the analysis of milk and dairy products ingredients. Recently, several methods based on polymerase chain reaction (PCR), including restriction fragment length polymorphism (PCR-RFLP), multiplex PCR, species-specific PCR, and real-time PCR, have been proposed as useful means for identifying species of origin in milk and dairy products, as well as quantifying and detecting any adulteration. These methods have particular advantages owing to their high specificity and sensitivity, as well as rapid processing time. In this review, we provide an updated and extensive overview of the PCR-based methods used for milk and dairy products authentication with a particular focus on the application of PCR methods to detect adulteration.

• Journal of Gastric Cancer
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• v.19 no.3
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• pp.235-253
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• 2019

Gastric cancer (GC) is one of the deadliest malignancies in the world. Currently, clinical treatment decisions are mostly made based on the extent of the tumor and its anatomy, such as tumor-node-metastasis staging. Recent advances in genome-wide molecular technology have enabled delineation of the molecular characteristics of GC. Based on this, efforts have been made to classify GC into molecular subtypes with distinct prognosis and therapeutic response. Simplified algorithms based on protein and RNA expressions have been proposed to reproduce the GC classification in the clinical field. Furthermore, a recent study established a single patient classifier (SPC) predicting the prognosis and chemotherapy response of resectable GC patients based on a 4-gene real-time polymerase chain reaction assay. GC patient stratification according to SPC will enable personalized therapeutic strategies in adjuvant settings. At the same time, patient-derived xenografts and patient-derived organoids are now emerging as novel preclinical models for the treatment of GC. These models recapitulate the complex features of the primary tumor, which is expected to facilitate both drug development and clinical therapeutic decision making. An integrated approach applying molecular patient stratification and patient-derived models in the clinical realm is considered a turning point in precision medicine in GC.

• Journal of Medicine and Life Science
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• v.20 no.3
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• pp.107-114
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• 2023

Epidemiological control of coronavirus disease 2019 (COVID-19) is needed to estimate the infection period of confirmed cases and identify potential cases. The present study, targeting confirmed cases for which the time of COVID-19 symptom onset was disclosed, aimed to investigate the relationship between intervals (day) from symptom onset to testing the cycle threshold (CT) values of real-time reverse transcription-polymerase chain reaction. Of the COVID-19 confirmed cases, those for which the date of suspected symptom onset in the epidemiological investigation was specifically disclosed were included in this study. Interval was defined as the number of days from symptom onset (as disclosed by the patient) to specimen collection for testing. A locally weighted regression smoothing (LOWESS) curve was applied, with intervals as explanatory variables and CT values (CTR for RdRp gene and CTE for E gene) as outcome variables. After finding its non-linear relationship, a polynomial regression model was applied to estimate the 95% confidence interval values of CTR and CTE by interval. The application of LOWESS in 331 patients identified a U-shaped curve relationship between the CTR and CTE values according to the number of interval days, and both CTR and CTE satisfied the quadratic model for interval days. Active application of these results to epidemiological investigations would minimize the chance of failing to identify individuals who are in contact with COVID-19 confirmed cases, thereby reducing the potential transmission of the virus to local communities.

• The Plant Pathology Journal
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• v.39 no.6
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• pp.614-624
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• 2023

Botrytis cinerea is a major fungal plant pathogen that causes gray mold disease in strawberries, leading to a decrease in strawberry yield. While benzimidazole is widely used as a fungicide for controlling this disease, the increasing prevalence of resistant populations to this fungicide undermines its effectiveness. To investigate benzimidazole resistant B. cinerea in South Korea, 78 strains were isolated from strawberries grown in 78 different farms in 2022, and their EC₅₀ values for benzimidazole were examined. As a result, 64 strains exhibited resistance to benzimidazole, and experimental tests using detached strawberry leaves and the plants in a greenhouse confirmed the reduced efficacy of benzimidazole to control these strains. The benzimidazole resistant strains identified in this study possessed two types of mutations, E198A or E198V, in the TUB2 gene. To detect these mutations, TaqMan probes were designed, enabling rapid identification of benzimidazole resistant B. cinerea in strawberry and tomato farms. This study utilizes TaqMan real-time polymerase chain reaction analysis to swiftly identify benzimidazole resistant B. cinerea, thereby offering the possibility of effective disease management by identifying optimum locations and time of application.

• Journal of the Korean Chemical Society
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• v.54 no.2
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• pp.215-221
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• 2010

Salmonella is an important food-and water-borne pathogen associated with acute gastrointestinal illnesses around the world. The most common serotypes isolated from humans are Salmonella enterica serotype Typhimurium (S. Typhimurium) and S. Enteritidis. Traditional detection methods for Salmonella are based on cultures using selective media and characterization of suspicious colonies by biochemical and serological tests. These methods are generally time-consuming and not so highly sensitive. Recently, the Loop Mediated Isothermal Amplification and real-time PCR has been used as a highly sensitive, specific, and rapid test for the presence of pathogenic bacteria. In this study, a LAMP and real-time PCR was used to detect S. Typhimurium and S. Enteritidis. We selected target genes, which were the in invA and a randomly cloned sequence specific for the genus Salmonella. With LAMP and real-time PCR, random sequence was detected from Salmonella spp, invA were detected from all strain of S. Typhimurium and S. Enteritidis. This assay indicate that the specificity, sensitivity and rapid of the LAMP and real-time PCR make them potentially valuable tools for detection of S. Typhimurium and S. Enteritidis.

• Journal of Microbiology and Biotechnology
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• v.24 no.8
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• pp.1026-1033
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• 2014

The fecal microbiotas were investigated in 13 healthy Thai subjects using polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE). Among the 186 DNA bands detected on the polyacrylamide gel, 37 bands were identified as representing 11 species: Bacteroides thetaiotaomicron, Bacteroides ovatus, Bacteroides uniformis, Bacteroides vulgatus, Clostridium colicanis, Eubacterium eligenes, E. rectale, Faecalibacterium prausnitzii, Megamonas funiformis, Prevotella copri, and Roseburia intestinalis, belonging mainly to the groups of Bacteroides, Prevotella, Clostridium, and F. prausnitzii. A dendrogram of the PCR-DGGE divided the subjects; vegetarians and non-vegetarians. The fecal microbiotas were also analyzed using a quantitative real-time PCR focused on Bacteroides, Bifidobacterium, Enterobacteriaceae, Clostrium coccoides-Eubacterium rectale, C. leptum, Lactobacillus, and Prevotella. The nonvegetarian and vegetarian subjects were found to have significant differences in the high abundance of the Bacteroides and Prevotella genera, respectively. No significant differences were found in the counts of Bifidabacterium, Enterobacteriaceae, C. coccoides-E. rectale group, C. leptum group, and Lactobacillus. Therefore, these findings on the microbiota of healthy Thais consuming different diets could provide helpful data for predicting the health of South East Asians with similar diets.