Detection of Rifampin Resistant Mycobacterium tuberculosis complex using Denaturing HPLC / 대한진단검사의학회지

BACKGROUND: Tuberculosis (TB) remains an important cause of morbidity and mortality throughout the world. The surge of TB has been accompanied by an increase in multi-drug-resistant tuberculosis (MDR-TB). In this study, we developed a denaturing HPLC (DHPLC) method for detecting rpoB gene mutation as a rifampin resistance based on sequence. METHODS: In this study, we used 99 mycobacterial isolates grown in Ogawa media. At first, we used a PCR method that can amplify the 235 bp and 136 bp rpoB DNAs of Mycobacterium tuberculosis complex (MTB) and Non-tuberculous mycobacteria (NTM). And then, PCR-restriction fragment length polymorphism (RFLP) of rpoB DNA (342 bp), which comprises the Rif(T) region, was used for the differential identification of Mycobacteria. Finally, we detected these amplicons by DHPLC, compared to PCR-RFLP results, and performed sequencing. RESULTS: Among 99 mycobacterial isolates, 80 (81%) were MTB and 19 (19%) were NTM. NTM were identified to 7 different species by DHPLC and PCR-RFLP. rpoB mutation was detected in 9 (11%) of the MTB specimens. These results were confirmed by using sequencing. CONCLUSIONS: DHPLC provided a rapid, simple, and automatable performance for detection of rifampin resistant Mycobacterium tuberculosis complex and would be helpful as a supplemental method in high-throughput clinical laboratories.

Quantitative Determination of Plasma and Urine Paraquat Concentrations using Spectroscopic Method / 대한진단검사의학회지

BACKGROUND: It is well known that plasma paraquat concentration is one of the most important prognostic indicators for paraquat poisoning. Quantitative analyses of paraquat, however, are not generally used in clinical laboratories. In this work, we evaluated the second-derivative spectroscop-ic method for quantitation of paraquat in plasma and urine, and investigated the clinical significance in patients with paraquat poisoning. METHODS: Linearity, precision, interferences, and comparison with high-performance liquid chro-matography (HPLC) were evaluated in 20 paraquat-poisoning cases using the UV-160 A recording spectrophotometer. The relationship of plasma and urine paraquat concentrations with the clinical outcomes was also studied. RESULTS: The within-run and between-day coefficients of variation (CV) for groups of low and high levels were less than 5%. The derivative amplitude was linearly related to paraquat concentra-tion through the range from 0.5 to 10 ng/mL. The correlation coefficient (r) between spectrophotom-etry and HPLC was 0.992. The accuracy for predicting the outcome for patients based on plasma paraquat concentration was 84.6%. The urine paraquat levels on admission were more than 10 ng/ mL in all of the 9 non-survivors group and in 5 out of 11 of the survivors group. The eliminating rates for plasma and urine paraquat concentrations by extracorporeal procedures were not statistically different between the two groups. CONCLUSIONS: Second-derivative spectroscopic methods for quantitation of paraquat showed an acceptable performance and suitable procedure for clinical laboratory use and it was thought to be seful in assessing the severity and in predicting the prognosis for paraquat poisoning.

Detecting p53 gene mutation of breast cancer and defining differences between silver staining PCR-SSCP and immunohistochemical staining

This study detects and defines the patterns of p53 gene mutations in breast cancers. We analyse p53 gene mutations through comparing the results of single-strand-conformation-polymorphism (SSCP) and immunohistochemistry (IHC), and we try to define the differences between the results of SSCP and IHC. Twenty-seven fresh primary breast cancer tissues and eight normal breast tissues were studied. The IHC was done with the usual streptavidin-biotin peroxidase complement method by using monoclonal antibody DO-7. The results of staining was scored. The SSCP method was done by using Cold SSCP Electrophoresis System. Overexpressions of p53 protein were seven (25.9%) among 27 cancer cases on IHC. Four (57.1%) of seven cases were positive in SSCP. In SSCP, the mutations were detected in 10 (37%) among 27 cancer cases. The mutations were two in exon 5, one in exon 8, and seven cases in exon 7. All of 10 mutations were proved by sequencing analysis. Of them, only four (40%) were positive in IHC. We consider the IHC as a screening method for p53 gene mutations.

Comparative effects on secretion of LH, FSH, prolactin, and testosterone by chronic and direct hypothalamic administration of nonylphenol to adult male rats

Nonylphenol (NP) is a widespread environmental pollutant that has been shown to exert both toxic and estrogenic effects on mammalian cells. As the effects of NP on the reproductive system of adult male vertebrates are virtually unknown, we investigated not only the changes of reproductive hormone secretion in serum after chronic exposure to NP but also, in order to identify the site of its action, the reproductive hormone secretion in serum 48 hours after microinfusion of NP within hypothalamic preoptic area (POA). In the chronic exposure, the luteinizing hormone (LH), follicle stimulating hormone (FSH), and testosterone in serum were decreased but prolactin (PRL) concentrations were increased. The LH, FSH, and testosterone in serum were decreased through the direct infusion of NP into POA, while there was no difference in mean serum prolactin between NP and control groups. These observations suggest that NP as endocrine disruptor has modulatory effects on hypothalamo-pituitary-gonadalaxis and that the site of action of NP could be hypothalamic POA.