Human Papillomaviruses(HPVs) are regarded as sexually transmitable and closely associated with precancerous lesions or invasive cancers of the vulva, vagina, and cer V1X. In situ hybridization, besides showing higher sensitivity, allowed the identification of specific viral types. We studied 12 cases of condylomata accuminata for HPV typing by in situ hybridization techenique. Ten cases(83.3 % ) showed positive for HPV type 6/11, and 2 cases(16.7% ) were positive for HPV type 16/18.
DNA Probes* ; DNA* ; Humans* ; In Situ Hybridization* ; Vagina ; Vulva

No abstract available.
DNA Fingerprinting* ; DNA* ; Oligonucleotide Probes* ; Polymerase Chain Reaction*

Breast Neoplasms ; genetics ; DNA Probes ; Female ; Genes, erbB-2 ; Humans

Vibrio splendidus is an opportunistic pathogen in aquaculture. It can infect a variety of aquaculture animals and has caused huge losses to the aquaculture industry. In this study, a novel and efficient method for detecting V. splendidus was developed by combining the exonuclease Ⅲ amplification strategy with a nucleic acid test strip developed based on gold nanoparticles-labeled DNA probe. The results could be directly visualized by naked eyes, and this system overcame the difficulty in preparation of the monoclonal antibody used in conventional immunostrip. Upon optimization of experimental conditions, the detection limit of the strip was 5 ng/mL for the synthetic oligonucleotide DNA fragment and 10 ng/mL for the actual genomic DNA sample of V. splendidus. This test strip was more sensitive compared with the PCR method and was specific for the detection of V. splendidus. The rapid preparation of nucleic acid strip and the efficient detection of V. splendidus open a new way for the prevention and control of aquatic diseases.
Animals ; DNA Probes ; Gold ; Metal Nanoparticles ; Vibrio/genetics*

Genomic DNAs were extracted from cervical lavages of 49 patients with cervical cancer. Dot and Southern blot hybridization were performed using the P-labeled HFV DNA probes to find high risk HPV(type 16 and 18) infection that is known as the mast prevalent pathogenic factor in cervical cancer. Furthermore, genornic DNAs purified frnm cervical cancer tissues were studied in 8 out of 49 patients allowing us to convince the results from cervical lavages. The results were as follaws: 1. Dot blot analysis were used to examine the sensitivity and specificity of hybridization condition and HPV-DNA probes. Fasitive signals were obtained even at the level of 10pg for HPV DNA, but no signals could he detected at the level of as much as 400pg for salmon sperm DNA. 2. Dot blot of DNAs from cervircal lavages showed positive signals in 32.7%(16/49) with HPV type 16 probe and 20.4% (10/49)and one mixed infection was found. 3. When the DNAs from cervircal lavages of 49 patients were classified according to the clinical stage of cervical cancer, the infection rates of HPV type 16 and 18 were 50% (2/4) in CIN, 80% (4/5) in stage I, 64. 2% (9/14) in stage I b, 45% (9I20) in stage II and 16. 7% (1/6) in stage Ill and K respectively. The occurrenr,e of HPV type 16 and 18 seemed to be the highest in the cervical cancer stage 1 (68.4%(13/19). 4. Experiments perfornecl with genomic DNAs from 8 cancer tissues showed similar results compared to those of cervical lavages, but the intensity of positive signals was stronger. 5. Genomic DNAs from 5 patients(3 cases from cervical lavages and 2 cases from cervical cancer tissues) which showed strong positive signals to the dot blot analysis were further examined by Southern blot hybridixation using HFV type 16 DNA probe. When DNAs were digested with Pst 1 restriction enzyme, the five characteristic frgmenta of BFV type 16(2.8, l.9, l.6, 1.0 and 0.5 kb long in length) were recognized in ell 5 cases, These results may suggest a direet relatianship between HPV type 16 & 18 infectioas considered as the most effective methods for HPV detectioe and typing. Mo1ecular biclogieal studies in the reserarch of HPV are expected to reveal and help us understand the pathogenesis of cervical cancer.
Blotting, Southern ; Coinfection ; DNA Probes ; DNA* ; Humans* ; Salmon ; Sensitivity and Specificity ; Spermatozoa ; Therapeutic Irrigation ; Uterine Cervical Neoplasms*

Eighty-two strains of Escherichia coli isolated from urine specimens in Pusan University Hospital, were serotyped and analyzed for plasmid DNA profiles, PFGE profiles, MRHA of human blood cells, HEp-2 cell adherence ability and reactivity to bfpA, LT, STh and STp DNA probes. The following results were obtained. Fifty-three of the eighty-two strains belonged to thirteen different 0 serotypes, twenty-nine strains could not be typed with the antisera used. Thirty strains (43.9%) were hemolysin producer. MRHA is present on twenty-nine strains (35.37%) of eighty-two strains. MRHA positive strains carry a plasmid of 60MDa, a putative factor involved in adherence. This plasmid might be specific for MRHA positive strains. MRHA positive strains were observed in serotype 01, 018, 055, 086a, 0119, 0126, and 0142. Twenty-six strains of E. coli showed three patterns of adherence to HEp-2 cells namely, localized, diffuse, and aggregative adhesion. Twenty-two strains hybridized with the bfpA probe, while all eighty-two strains did not hybridize with the probes, LT, STh, STp. The restriction fragment patterns of chromosomal DNA digested with AotI analysed by PFGE of hemolysin-producing E. coli ten strains were compared with eight different types. Three of E. coli serotype 01, 08 and 0126 showed the same chromosomal DNA fragment patterns.
Blood Cells ; Busan ; DNA ; DNA Probes ; Escherichia coli* ; Escherichia* ; Humans ; Immune Sera ; Plasmids ; Serotyping*

The objective of this study was to develop PCR primers that are specific for Streptococcus sanguinis, Streptococcus parasanguinis, and Streptococcus gordonii. We designed the S. sanguinis-, S. parasanguinis-, and S. gordonii-specific primers, Ssa21-F3/Ssa21-R2, Spa17-F/Spa17-R, and Sgo41-F1/Sgo41-R1 respectively, based on the nucleotide sequences of the Ssa21, Spa17, and Sgo41 DNA probes that were screened using inverted dot blot hybridization (IDBH). The species-specificity of these primers was assessed against 43 strains of mitis group streptococci, including clinical strains of S. sanguinis, S. parasanguinis, and S. gordonii. The resulting PCR data revealed that species-specific amplicons had been obtained from all strains of the target species tested, and that none of these amplicons occurred in any other strains from other species. These results suggest that the Ssa21-F3/Ssa21-R2, Spa17-F/Spa17-R, and Sgo41-F1/Sgo41-R1 primers may be useful in detecting S. sanguinis, S. parasanguinis, and S. gordonii at the species level, respectively.
Base Sequence ; Chimera ; DNA ; DNA Probes ; Polymerase Chain Reaction ; Streptococcus ; Streptococcus gordonii

In this study, the specific primers and probes of Panax quinquefolius were designed for a quantitative real-time PCR, and the rapid identification method of P. quinquefolius was established by optimizing conditions. The method was used to validate 43 samples of the traditional Chinese medicine,and the results showed that 22 samples of P. quinquefolius were identified accurately. The limit of detection of the method can be reach to 1×10⁻⁴ ng. The method is accurate, fast, sensitive and specifically.
DNA Primers ; DNA Probes ; Drugs, Chinese Herbal ; standards ; Panax ; genetics ; Real-Time Polymerase Chain Reaction

OBJECTIVE: To develop a simple, sensitive and robust method for rapid detection of human apurinic/apyrimidinic endonuclease 1 (APE1) in various biological samples. METHODS: An abasic site-containing DNA probe with a sequence of 5'-T*T*C*C*T*C*T(ROX)AGAGXCGTT (BHQ2)C*A*C*T*G*T*AGTTTATA*C*A*G*T*GAATCTCTCTAG*T*C*T-3' ["X" represents AP site; The phosphorothioated nucleotides (at 3' side) are indicated with an asterisk after the nucleotides; ROX is 6-carboxy-X-rhodamine and BHQ2 is Black Hole quencher 2] was synthesized and used for the detection. In the presence of APE1, the DNA probe could be specifically hydrolyzed by the enzyme and release the fluorophore, resulting in strong fluorescence emission. The activity of APE1 was determined according to the rate of increase in fluorescence intensity. In this work, we modified the reaction buffer and significantly improved the performance of the method. Moreover, the method was further extended to measure the contents of APE1 in the protein extraction from peripheral blood mononuclear cells (PBMCs) extracted from human whole blood samples by density gradient centrifugation. The assay was also applied to measure the activity of APE1 in human serum samples. RESULTS: With a new reaction buffer composed of 0.04% (V/V) Triton X-100, 50 mmol/L KAc, 20 mmol/L Tris-Ac, 10 mmol/L Mg(Ac)2 and 1 mmol/L dithiothreitol (DTT), the method achieved a detection limit of 0.005 U/mL (3 pg/mL) and a linear response ranging from 6 pg/mL to 1.2 ng/mL. The contents of APE1 in the protein extraction from PBMCs of eight blood samples were measured to be in the range from 0.061 to 0.40 ng/μg protein, with an average of 0.16 ng/μg protein. The recovery was 98%±5% (n=3). The levels of APE1 in the sera from 102 normal individuals (51 male and 51 female, age range: 59-75 years) were observed to be from 0.13 to 0.34 ng/mL, with a recovery of 96%±15% (n=3). CONCLUSION The new fluorescence assay was simple, rapid and sensitive, providing a practical tool to measure the activity of APE1 in serum samples and cell extracts. It also holds great potential in measurement of APE1 in many other biological samples for clinical test and laboratory research.
Aged ; DNA Probes ; DNA-(Apurinic or Apyrimidinic Site) Lyase ; Female ; Fluorescence ; Humans ; Leukocytes, Mononuclear ; Male ; Middle Aged

BACKGROUND: The conventional method for decalcification of bone specimens uses hydrochloric acid (HCl) and is notorious for damaging cellular RNA, DNA, and proteins, thus complicating molecular and immunohistochemical analyses. A method that can effectively decalcify while preserving genetic material is necessary. METHODS: Pairs of bilateral bone marrow biopsies sampled from 53 patients were decalcified according to protocols of two comparison groups: EDTA versus HCl and RDO GOLD (RDO) versus HCl. Pairs of right and left bone marrow biopsy samples harvested from 28 cases were allocated into the EDTA versus HCl comparison group, and 25 cases to the RDO versus HCl comparison group. The decalcification protocols were compared with regards to histomorphology, immunohistochemistry, and molecular analysis. For molecular analysis, we randomly selected 5 cases from the EDTA versus HCl and RDO versus HCl groups. RESULTS: The decalcification time for appropriate histomorphologic analysis was the longest in the EDTA method and the shortest in the RDO method. EDTA was superior to RDO or HCl in DNA yield and integrity, assessed via DNA extraction, polymerase chain reaction, and silver in situ hybridization using DNA probes. The EDTA method maintained intact nuclear protein staining on immunohistochemistry, while the HCl method produced poor quality images. Staining after the RDO method had equivocal results. RNA in situ hybridization using kappa and lambda RNA probes measured RNA integrity; the EDTA and RDO method had the best quality, followed by HCl. CONCLUSIONS: The EDTA protocol would be the best in preserving genetic material. RDO may be an acceptable alternative when rapid decalcification is necessary.
Biopsy* ; Bone Marrow* ; Decalcification Technique ; DNA ; DNA Probes ; Edetic Acid ; Humans ; Hydrochloric Acid ; Immunohistochemistry ; In Situ Hybridization ; Nuclear Proteins ; Polymerase Chain Reaction ; RNA ; RNA Probes ; Silver