Methods Cytological smears were prepared from urinary sediment obtained on the third, fifth, and 15th days. The smears were fixed in 95% ethanol and stained by the Papanicolaou method.The third day urinary sediment was examined electron microscopically. The cell pellet was fixed with 2-5% glutaraldehyde and 1% osmic acid, dehydrated with graded alcohol, and embedded in Polybed 812. Ultrathin sections were made, stained with uranyl acetate and lead citrate, and observed with an electron microscope (H-7000, Hitachi, Japan).About 200-300 abnormal urothelial cells on a glass slide prepared by the Papanicolaou method were used for DNA extraction. The Papanicolaou stained smear was immersed in xylene in a 60°C incubator for one day, after which the cover glass was detached. Xylene was removed with alcohol. After washing with distilled water, digestion of the protein of the urothelial cells was performed on the glass slide in 300 ,ul proteinase K solution (200,ug/ml) at 37°C for 18 hours. The DNA was then extracted with phenol. Extractions with chloroform were successively repeated three times and the phenol was eliminated. Next, the RNA in the sample was digested with 100 ug/ml RNase at 37°C for 15 minutes and the DNA was again extracted with phenol. Extractions with chloroform were then repeated three times and the RNase was completely eliminated. The DNA sample was finally precipitated with ethanol and dissolved in 50 pul of 10 mM TRIS-HCl buffer (pH 7 5) containing 1 mM EDTA. The sample was frozen and thawed several times before PCR to destroy inhibitors of Taq polymerase in the urne.7The sample DNA, the sample DNA diluted three times with phosphate buffered saline (PBS), DNAs of BK and JC viruses used as positive controls and human placental DNA used as a negative control, and PBS without DNA were prepared for amplification by polymerase chain reaction (PCR). The control BK virus DNA used had been extracted from the urine of a patient with a urinary bladder carcinoma and was found to be positive for BK virus using PCR. The JC virus DNA was extracted from the brain tis-773 on 11 May 2018 by guest. Protected by copyright.
We report a familial clustering case of hepatitis delta. All of the members of this family had evidence of past infection of hepatitis B. We investigated the hepatitis delta, three of the members had positive serological hepatitis delta markers. We assayed by polymerase chain reaction the primers corresponding to hepatitis delta antigen. The results of polymerase chain reaction was three positive. The 2nd polymerase chain reaction was used, two geno-type specific primers one was for Japanese S type the other was for Japanese M type7). Three were positive the 2nd polymerase chain reaction for Japanese M, one was negative for all of hepatitis delta polymerase chain reaction.
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