• Journal of Microbiology and Biotechnology
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• 제30권4호
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• pp.533-539
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• 2020

A quantitative real-time polymerase chain reaction (QPCR) was applied to estimate biokinetic coefficients of Clostridium cadaveris and Clostridium sporogenes, which utilize protein as carbon source. Experimental data on changes in peptone concentration and 16S rRNA gene copy numbers of C. cadaveris and C. sporogenes were fitted to model. The fourth-order Runge-Kutta approximation with non-linear least squares analysis was employed to solve the ordinary differential equations to estimate biokinetic coefficients. The maximum specific growth rate (μ_max), half-saturation concentration (K_s), growth yield (Y), and decay coefficient (K_d) of C. cadaveris and C.sporogenes were 0.73 ± 0.05 and 1.35 ± 0.32 h^-1, 6.07 ± 1.52 and 5.67 ± 1.53 g/l, 2.25 ± 0.75 × 10¹⁰ and 7.92 ± 3.71 × 10⁹ copies/g, 0.002 ± 0.003 and 0.002 ± 0.001 h^-1, respectively. The theoretical specific growth rate of C. sporogenes always exceeded that of C. cadaveris at peptone concentration higher than 3.62 g/l. When the influent peptone concentration was 5.0 g/l, the concentration of C.cadaveris gradually decreased to the steady value of 2.9 × 10¹⁰ copies/ml at 4 h Hydraulic retention time (HRT), which indicates a 67.1% reduction of the initial population, but the wash out occurred at HRTs of 1.9 and 3.2 h. The 16S rRNA gene copy numbers of C. sporogenes gradually decreased to steady values ranging from 1.1 × 10¹⁰ to 2.9 × 10¹⁰ copies/ml. C. sporogenes species was predicted to wash out at an HRT of 1.6 h.

• Asian-Australasian Journal of Animal Sciences
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• 제28권11호
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• pp.1649-1656
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• 2015

Gene expression profiling has offered new insights into postmortem molecular changes associated with meat quality. To acquire reliable transcript quantification, high quality RNA is required. The objective of this study was to analyze integrity of RNA isolated from chicken skeletal muscle (pectoralis major) and its capability of serving as the template in quantitative real-time polymerase chain reaction (qPCR) as a function of postmortem intervals representing the end-points of evisceration, carcass chilling and aging stages in chicken abattoirs. Chicken breast muscle was dissected from the carcasses (n = 6) immediately after evisceration, and one-third of each sample was instantly snap-frozen and labeled as 20 min postmortem. The remaining muscle was stored on ice until the next rounds of sample collection (1.5 h and 6 h postmortem). The delayed postmortem duration did not significantly affect $A_{260}/A_{280}$ and $A_{260}/A_{230}$ ($p{\geq}0.05$), suggesting no altered purity of total RNA. Apart from a slight decrease in the 28s:18s ribosomal RNA ratio in 1.5 h samples (p<0.05), the value was not statistically different between 20 min and 6 h samples ($p{\geq}0.05$), indicating intact total RNA up to 6 h. Abundance of reference genes encoding beta-actin (ACTB), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), hypoxanthine-guanine phosphoribosyltransferase (HPRT), peptidylprolylisomerase A (PPIA) and TATA box-binding protein (TBP) as well as meat-quality associated genes (insulin-like growth factor 1 (IGF1), pyruvate dehydrogenase kinase isozyme 4 (PDK4), and peroxisome proliferator-activated receptor delta (PPARD) were investigated using qPCR. Transcript abundances of ACTB, GAPDH, HPRT, and PPIA were significantly different among all postmortem time points (p<0.05). Transcript levels of PDK4 and PPARD were significantly reduced in the 6 h samples (p<0.05). The findings suggest an adverse effect of a prolonged postmortem duration on reliability of transcript quantification in chicken skeletal muscle. For the best RNA quality, chicken skeletal muscle should be immediately collected after evisceration or within 20 min postmortem, and rapidly preserved by deep freezing.

• 대한환경공학회지
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• 제32권2호
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• pp.121-130
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• 2010

산성광산배수(Acid Mine Drainage; AMD)는 낮은 pH조건에서 중금속 및 황산염이온 등이 다량 용존되어 환경오염 문제를 발생시킨다. 국내의 폐광산 일부에서는 산성광산배수를 처리하기 위해 정화시설이 운영되고 있으나 여전히 주변 하천에 영향을 미치고 있다. 본 연구는 산성광산배수 및 영향을 받는 하천에서 지표미생물의 특이적 유전자를 실시간 정량 중합효소 연쇄반응(Real-time quantitative Polymerase Chain Reaction; Real-time qPCR)을 이용하여 확인 및 정량함으로써 광산배수의 환경영향을 미생물학적으로 판단하고자 수행되었다. 지표 종으로 선정한 미생물은 16S rRNA 미생물 군집분석 결과 발견된 미생물 중 철환원균인 Rhodoferax ferrireducens T118, Acidiphilium cryptum JF-5이며 이 외에 기존에 광산에 존재하는 것으로 알려진 미생물 중 호산성 황환원균인 Desulfosporosinus orientus, 철산화균인 Leptosprillum ferrooxidans, 철 및 황산화균인 Acidothiobacillus ferrooxidans이었다. 최종적으로, 본 연구에서 각 광산의 광산배수가 하천에 미치는 영향을 정량적으로 판단하여 비교하기 위해 광산배수로 인한 하천에서의 미생물 변동 지수를 산정하였으며 연구 대상 4개 광산 중 광산배수 처리시설이 없는 삼탄의 광산배수의 경우 주변 방류 하천으로의 미생물학적 환경영향이 가장 큰것으로 나타났다

• Asian Pacific Journal of Cancer Prevention
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• 제17권5호
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• pp.2705-2710
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• 2016

Recent evidence haas indicated that meningioma-associate protein (MAC30) exhibits different expression patterns in various tumors. However, little is known about the value of MAC30 in human squamous cell carcinoma of lung (SQCLC). The purpose of our study was to investigate the expression of MAC30 and to explore its clinical significance in SQCLC patients. A total of 156 Chinese patients diagnosed with SQCLC were selected for this study. The expression of MAC30 in all tissues was confirmed by immunohistochemical staining. Quantitative real-time PCR was performed to analyze MAC30 mRNA expression in 32 cases of SQCLC patients with corresponding non-tumor lung tissues. We observed enhanced mRNA expression of MAC30 in SQCLC as compared to control samples. Further, elevated MAC30 protein expression was strongly associated with poor tumor differentiation, TNM stage, and lymph node metastasis. In addition, we observed that patients with increased MAC30 expression demonstrated poor overall survival. Multivariate analysis explicated that increased MAC30 expression was a valuable independent predictable factor for poor tumor differentiation and short survival in SQCLC patients. Our present study suggests that MAC30 may serve as a biomarker for poor tumor differentiation and outcomes of patients with SQCLC.

• 대한의생명과학회지
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• 제19권2호
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• pp.90-97
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• 2013

The tuberculin skin test (TST) and interferon gamma (IFN-${\gamma}$) release assay (IGRA) have been widely used for diagnosis of latent tuberculosis infection (LTBI). In order to overcome limitations of current LTBI diagnostic methods, the development of a novel molecular assay which is able to measure the IFN-${\gamma}$ messenger RNA (mRNA) expression level after stimulation with Mycobacterium tuberculosis (MTB) specific antigen was recently developed. The ability of a molecular assay to detect MTB infection was similar to commercial IGRA however, the optimal incubation time for stimulating IFN-${\gamma}$ was not yet established. Therefore, in this study the direct comparisons of MTB Ag stimulation times (4 and 24 hrs) were performed for diagnosis of MTB infection. Data showed that the coincident rate between QFT-GIT IFN-${\gamma}$ ELISA and IFN-${\gamma}$ RT-PCR (4 hrs) was 88.35% and that of QFT-GIT and IFN-${\gamma}$ RT-PCR (24 hrs) was 70.85%. Based on a receiver operating characteristic (ROC) curve, the 4 hrs-MTB specific Ag stimulation time for IFN-${\gamma}$ RT-PCR had the significant P value, 95% CI value, and AUC (P < 0.0001, 95% CI=0.82 to 1.02, and AUC=0.9214) in comparison with 24 hrs-MTB specific Ag stimulation time (P = 0.009, 95% CI=0.06 to 0.94, and AUC=0.7711). These results show that 4-hr was the most optimal MTB Ag stimulation time for performing IFN-${\gamma}$ RT-PCR. Although semi-quantitative RT-PCR had a few analytical limitations, it might be useful as an alternative molecular diagnostic method for detecting MTB infection.

• Animal Bioscience
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• 제34권12호
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• pp.1886-1894
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• 2021

Objective: Growth hormone (GH) and insulin-like growth factor I (IGF-I) play a critical role in animal growth rates. We aimed to investigate the effect of GH and IGF-I genotypes on body weight (BW), dominance, and gene expression in slow-growing chickens at different ages. Methods: A total of 613 Korat chickens (KRs) were bred and divided into three groups by genotype - A1A1, A1A3, and A3A3 for GH and AA, AC, and CC for IGF-I. Chickens were weighed every two weeks, and liver and breast muscle tissues were collected at 10 weeks of age. Genetic parameters of KRs were estimated using ASReml software. The GH and IGF-I mRNA levels were measured by quantitative polymerase chain reaction. Significant differences between traits were analyzed using the generalized linear model. Results: A significant effect of GH genotypes on BW was found at most ages, and the A1A1 genotype had the highest value of BW. Compared with the A3A3 genotype, the A1A1 and A1A3 genotypes showed a higher dominance effect at 0 and 2 weeks, and genotype A1A1 had the highest value of dominance at 8 weeks of age. A difference in GH mRNA levels between genotypes was detected in breast muscle at 6 weeks and in the liver tissue at 2 weeks. In the case of IGF-I gene, the AA genotype had the highest BW at the beginning of life. Significant differences in BW dominance were found at 2 weeks. However, IGF-I mRNA levels were not different among genotypes in both breast muscles and liver tissues. Conclusion: Our results revealed that GH and IGF-I influence growth, but may not be involved in heterosis. GH can be used as a marker gene in selection programs for growth because the homozygous genotype (A1A1) had the highest BW at all ages. The IGF-I is not a useful marker gene for selection programs.

• Asian-Australasian Journal of Animal Sciences
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• 제29권3호
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• pp.343-351
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• 2016

Although the chemical, physical, and nutritional properties of bovine milk have been extensively studied, only a few studies have attempted to characterize milk-synthesizing genes using RNA-seq data. RNA-seq data was collected from 21 Holstein samples, along with group information about milk production ability; milk yield; and protein, fat, and solid contents. Meta-analysis was employed in order to generally characterize genes related to milk production. In addition, we attempted to investigate the relationship between milk related traits, parity, and lactation period. We observed that milk fat is highly correlated with lactation period; this result indicates that this effect should be considered in the model in order to accurately detect milk production related genes. By employing our developed model, 271 genes were significantly (false discovery rate [FDR] adjusted p-value<0.1) detected as milk production related differentially expressed genes. Of these genes, five (albumin, nitric oxide synthase 3, RNA-binding region (RNP1, RRM) containing 3, secreted and transmembrane 1, and serine palmitoyltransferase, small subunit B) were technically validated using quantitative real-time polymerase chain reaction (qRT-PCR) in order to check the accuracy of RNA-seq analysis. Finally, 83 gene ontology biological processes including several blood vessel and mammary gland development related terms, were significantly detected using DAVID gene-set enrichment analysis. From these results, we observed that detected milk production related genes are highly enriched in the circulation system process and mammary gland related biological functions. In addition, we observed that detected genes including caveolin 1, mammary serum amyloid A3.2, lingual antimicrobial peptide, cathelicidin 4 (CATHL4), cathelicidin 6 (CATHL6) have been reported in other species as milk production related gene. For this reason, we concluded that our detected 271 genes would be strong candidates for determining milk production.

• Restorative Dentistry and Endodontics
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• 제30권5호
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• pp.372-384
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• 2005

이 연구에서는 Prevotella nigrescens (P. nigrescens)의 lipopolysaccharide (LPS)로 자극한 치주인대 섬유아세포에서 matrix metalloproteinase (MMP)와 tissue inhibitor of metalloproteinase (TIMP)의 생성 양상과, LPS를 수산화칼슘으로 처리했을 때의 영향을 평가하였다. P. nigrescens에서 추출, 정제한 여러 농도의 LPS와 수산화칼슘으로 처리한 LPS로 치주인대 섬유아세포를 자극하여, Immunoprecipitation법으로 MMP-1, -2, TIMP-1의 단백질 생성 양상을, real-time polymerase chain reaction법으로 MMP-1의 mRNA 발현 양상을 분석하였다. 이 연구의 결과는 아래와 같다. 1. MMP-1은 단백질과 유전자 수준 모두 자극 시간과 비례하여 증가하여 48시간에 최대값을 보였다. 2. MMP-2단백질 생성은 1, 10 mg/ml에서 자극 시간과 비례하여 증가하였다. 3. TIMP-1 단백질 생성은 24시간까지 증가하다가 48시간에 감소하였고, 0.1과 1 ${\mg}g/ml$에서 증가하였으나 10 ${\mu}g/ml$ 에서 억제되었다. 4. P. nigrescens의 LPS를 수산화칼슘으로 처리시 MMP-1의 mRNA 발현은 현저하게 감소하였다.

• Animal Bioscience
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• 제34권4호
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• pp.743-750
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• 2021

Objective: The objectives of this study were to explore the expression patterns of myosin heavy chain (MyHC) genes of different skeletal muscles from Chinese cattle, and to investigate the relationship between myofiber characteristics and meat quality of M. longissimus lumborum (LL), M. psoas major (PM), and M. semimembranosus (SM) from Chinese Luxi and Qinchuan cattle. Methods: Three major muscles including LL, PM, and SM from Chinese Luxi cattle and Chinese Qinchuan cattle were used in this study. The myofiber characteristics were measured by histochemical analysis. The MyHC isoforms expression was evaluated by real-time quantitative polymerase chain reaction. Quality traits including pH value, meat color, cooking loss, Warner-Bratzler shear force (WBSF) and sarcomere length were determined at day 5 postmortem. Results: PM muscle had higher pH value, a* value, sarcomere length and lower WBSF value compared to LL and SM muscles (p<0.05). Numbers of type I myofiber and the relative expression of MyHC I mRNA in PM muscle were higher than those of LL and SM muscles (p<0.05). Myofiber diameter of PM muscle was lower than that of LL and SM muscles, regardless of myofiber types (p<0.05). Conclusion: According to the stepwise linear regression analyses, tenderness was influenced by myofiber characteristics in all three examined muscles. Tenderness of beef muscles from Qinchuan and Luxi cattle could be improved by increasing numbers of type I myofiber.

• 생명과학회지
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• 제20권12호
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• pp.1896-1901
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• 2010

본 연구는 가축생균제용 유산균을 Real-time PCR정량분석법을 이용하여 분석하였다. SYBR Green1 방법과 Probe 방법을 이용하여 표준곡선을 제작한 결과, SYBR Green1 방법에서는 Slope 값이 -3.346이었고, Y절편은 33.18, $R^2$ 값은 0.993으로 나타났으며, Probe 방법에서는 Slope값이 -3.321이었고, Y절편은 39.10, $R^2$ 값은 0.995로 나타나, 이를 이용한 표준곡선 제작이 가능함을 알 수 있었다. SYBR Green1 방법을 이용한 생균제의 Lactobacilli 정성 정량 분석결과 Real-time PCR값은 4.46~6.56 log copies로 나타났고, 생균수 측정 결과 값은 5.63~7.59 log CFU/g로 나타났으며, Probe 방법을 이용한 생균제의 Lactobacilli 정성 정량 분석결과에서는 Real-time PCR 값은 5.51~7.00 log copies로 나타났으며, 생균수 측정 결과 값은 5.63~7.59 log CFU/g로 나타났다. 본 연구에서 실시한 RT PCR법은 3~4일이 소요되는 기존의 배지법과 비교하여 24시간 이내에 신속하게 검출이 가능하다고 여겨지며, 또한 RT PCR을 이용한 분석방법에서도 dye 사용과 primer 사용에 따라 결과값이 차이가 나타났음을 확인할 수 있었으며, Probe 방법을 이용하여 실험 한 결과가 민감한 결과를 나타내었음을 확인 할 수 있었다.