Masaharu Yokota

Effects of heparin on polymerase chain reaction for blood white cells

Polymerase chain reaction (PCR) has been used with increasing frequency to diagnose infectious and genetic diseases. In this study, the effects of heparin on PCR were investigated, because heparinized blood may sometimes be used in PCR studies. HLA‐DQA1 gene amplification was used as a model. PCR was clearly interfered with when heparinized blood was used as a source of template DNA, and the degree of interference was affected by the following three factors; (1) type of Taq DNA polymerase; (2) leukocyte count in blood; and (3) concentration of heparin contained. When additional tests were conducted with additions of definite heparin concentrations to a PCR reaction mixture, specimens with large amounts of DNA tended to exhibit less interference by heparin. The addition of ≥ 0.1 to 0.0016 U of heparin per reaction mixture (50 μl) suppressed DNA amplification in a dose‐dependent fashion. We therefore concluded that much care should be taken when heparinized blood is used as a PCR material. J. Clin. Lab. Anal. 13:133–140, 1999.

DNA extraction from human urinary sediment

DNA was extracted from urinary sediments and was sufficient for polymerase chain reaction (PCR) and enzymatic analysis, even if DNA from microorganisms coexisted. From urine samples, the yield of DNA ranged from trace levels to 20 μg per 10 mL urine. When urinary sediment was stored in ethanol, DNA remained stable for 2 weeks or more. Individual identification and sex determination could easily be performed using either fresh or ethanol‐fixed urine. In conclusion, urine can be used as a source for PCR‐based investigations and genetic studies. J. Clin. Lab. Anal. 12:88–91, 1998.

Bactericidal effects of konjac fluid on several food-poisoning bacteria.

In this study, the bactericidal effects of Japanese alkaline foods on food-poisoning bacteria were evaluated. Konjac is an alkaline food soaked in calcinated calcium (the pH of konjac fluid ranges from 11.42 to 12.53). Konjac fluids completely inactivated Escherichia coli, enterohemorrhagic E. coli O157:H7 and E. coil O26:H9, Salmonella Enteritidis, Vibrio parahemolyticus. and Staphylococcus aureus. The initial level of 6 log CFU/ml dramatically decreased after incubation with konjac fluid, and no viable gram-negative bacterium cells could be detected within 1 to 2 days and no viable S. aureus cells could be detected within 3 to 5 days. On the other hand, treatment with konjac fluid was also effective in reducing levels of spore-forming bacteria (Bacillus subtilis, Bacillus cereus, Clostridium perfringens, and Clostridium botulinum type E and type A). At least a 4-log reduction of spore-forming bacteria was obtained in konjac fluid within 7 to 14 days. Vegetative cells were more susceptible to konjac fluid than spores were. When the initial cell count was 6 log CFU/ml, a few surviving spores remained for 60 to 90 days, but no spores could be detected after 120 days. When the initial count of spore-forming bacteria was 3 to 4 log CFU/ml, the cells considered vegetative were completely inactivated within I to 3 days. Repeated treatment with konjac fluid caused complete inactivation of spores in less than 1 to 3 days. Our studies indicate that konjac fluid, which has a long history of use in food, will control food-poisoning bacterial contamination during the production or preservation of konjac and other foods and has a preventive effect on bacteria that can cause severe disease at uniquely low levels.

Tissue factor pathway inhibitor can interact with platelets.

Tissue factor pathway inhibitor (TFPI) regulates the extrinsic pathway of blood coagulation. However, there is no report on interaction between TFPI and platelets other than that by Tsuji, who found that whole blood anticoagulated with TFPI exhibited remarkable decrease in platelet count. Our study revealed that washed platelets suspended in modified Tyrodes buffer (8 mM CaCl2) containing TFPI exhibit platelet aggregation. However, platelets aggregation was observed without TFPI, but its increase and intensity were slow and weak, compared to that in the presence of TFPI. This aggregation was inhibited by anti-CD41 (anti-GPIIb) antibody. This finding suggested that TFPI promotes platelet aggregation.

A convenient method of DNA extraction from blood anticoagulated wity EDTA

Surplus blood often remains after routine clinical tests using EDTA‐anticoagulated samples. To use this blood for DNA analysis, we isolated white cells by adherence to polyethylene terephthalate fibers, which could be stored for several weeks transported by mail after methanol fixation. DNA yield was sufficient and correlated with white cell count. Extracted DNA was free of hemoglobin contamination and durable to polymerase chain reaction and enzyme digestion, which yielded products visualized as well‐separated bands on electrophoresis. We found our method to be practical for the routine clinical laboratory.

Survival of Serratia marcescens under dry condition isolated from nosocomial infection

平成 12 年 6 月,市内医療機関にて Serratia marcescens(以下セラチア)による院内感染事例が あり,当該病院内のふきとり検査から異なるセラ チアが病棟間で分離された.一般的にセラチア は水回りを中心に生存していると考えられてい る.しかし,院内伝播が不明確な今回の事例につ いて,感染経路を究明する一手段として,セラチ アが乾燥した状態で生存し得るか,生存すればそ の期間はどれぐらいであるのかを細菌学的に検討 した. 材料及び方法 前述の事例より分離された患者由来株 2株(No. 1,2),環境由来株 2株(No. 3,4)及び医療従事者 由来株 2株(No. 5,6)計 6株をトリプトソイブイ ヨン培地にて一夜培養し,滅菌生理食塩水で段階 希釈後,その希釈液 0.5ml(接種菌量 1.8~5.5× 10)を深型シャ-レに滴下,その後 25°Cの孵卵器 内(湿度 13%)にて乾燥試験を行った.48 時間後 には乾燥が完了しセラチアが白濁のスポットとし てシャーレ底にみられた.それらを 6カ月まで 2 週間毎に取り出し,そのシャーレにトリプトソイ ブイヨン培地 15ml を接種し 24 時間培養した.そ の後DHL寒天培地で培養し,セラチアの生存の 有無を確認した.また生存株については乾燥試験 前後のパルスフィ-ルド・ゲル電気泳動(以下 PFGE法)による遺伝子学的解析及びバイオテス ト 1号(栄研化学)を使用した生化学的性状を比 較検討した. 結果及び考察 長期乾燥試験の結果,患者由来株 2株,環境及 院内感染より分離したセラチアの乾燥生存試験

Availability of Nagarse for DNA analysis as a substitute for proteinase K

The availability of Nagarse, a protease, as a substitute for proteinase K for digestion of leukocytic or bacterial DNAs was studied. The amount and purity of DNAs extracted from leukocytes and several bacterial strains with Nagarse were compared with those of DNAs treated with proteinase. Nagarse exhibited the same behavior as proteinase K in digesting leukocytes, and it could also be used for bacterial digestion for physical mapping of genomic DNA by biased sinusoid field gel electrophoresis. Nagarse was thus comparable to proteinase K for use in biochemical experiments. The principal advantage of Nagarse is that it is inexpensive, unlike proteinase K, and our findings indicated that Nagarse is very useful as a substitute for proteinase K for the DNA study. J. Clin. Lab. Anal. 14:97–100, 2000.© 2000 Wiley‐Liss, Inc.

[A study on hemolytic streptococci (group A, B, C and G) isolated from throat of the middle-aged and advanced-aged--especially as compared with elementary school children].

Hemolytic streptococci were isolated from throat of middle-aged and advanced-aged and these organisms were classified into groups A, B, C and G. 1) Persons 15 to 39 years old were included in one group and persons from 40 years of age upward were divided into 5 groups every 10 years. Comparison of these groups were done. Generally, group B organisms were most often isolated. The detection rate of group B organisms was higher in the older age group. The detection rates of group A and G organisms were approximately equal, but both organisms were isolated from a few persons. No organism of group C was isolated from males, while only two strains belonged to group C organism were isolated from females. 2) A number of strains of group A hemolytic streptococci were isolated from school children, particularly in the lower classes. But the proportion of group B organism to isolated streptococci showed an increase in upper classes, noticeably in females. 3) For middle-aged and advanced-aged, blood samples were obtained simultaneously on examination of the throat, and ASO value and ASK titer were determined. ASO values were higher in persons infected with groups A, C or G organisms than in persons infected with group B organism or no hemolytic streptococcus. ASK titers revealed a similar results to ASO values, though the relation between ASK titers and isolated hemolytic streptococci was less positive.