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*

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

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

BACKGROUND: Intrachromosomal amplification of chromosome 21 (iAMP21) is known to be associated with poor prognosis in B-cell ALL (B-ALL). To determine the frequency and clinical characteristics of iAMP21 in Korean B-ALL patients, we performed FISH and multiplex ligation-dependent probe amplification (MLPA) analyses. METHODS: A total of 102 childhood B-ALL patients were screened with ETV6-RUNX1 FISH probes (Abbott Molecular, USA). The presence of an iAMP21 was confirmed by using MLPA P327 iAMP21-ERG probemix (MRC Holland, The Netherlands). RESULTS: iAMP21 was detected in one of the screened B-ALL patients (1/102 patients, 1.0%) who presented the ALL immunophenotype and complex karyotype at initial diagnosis. The patient relapsed twice after bone marrow transplantation. MLPA showed 12.5-Mb and 4.28-Mb regions of amplification and deletion, respectively. CONCLUSIONS: The frequency of iAMP21 is considerable in Korean pediatric patients. Our report suggests that iAMP21 in childhood B-ALL has very unfavorable impact on patient's prognosis. Additional methods such as MLPA analysis is essential to rule out patients with equivocal interphase FISH results.
Adolescent ; Asian Continental Ancestry Group/*genetics ; B-Lymphocytes/*metabolism ; Child ; Child, Preschool ; *Chromosomes, Human, Pair 21 ; Core Binding Factor Alpha 2 Subunit/genetics ; DNA Probes/metabolism ; Female ; Humans ; Immunophenotyping ; In Situ Hybridization, Fluorescence ; Infant ; Infant, Newborn ; Male ; Multiplex Polymerase Chain Reaction ; Precursor Cell Lymphoblastic Leukemia-Lymphoma/*diagnosis/genetics ; Proto-Oncogene Proteins c-ets/genetics ; Repressor Proteins/genetics ; Republic of Korea ; Translocation, Genetic ; Young Adult

BACKGROUND: Peptide nucleic acid (PNA) probes are artificial DNA analogues with a hydrophobic nature that can penetrate the mycobacterial cell wall. We evaluated a FISH method for simultaneous detection and identification of Mycobacterium tuberculosis (MTB) and non-tuberculous mycobacteria (NTM) in clinical respiratory specimens using differentially labeled PNA probes. METHODS: PNA probes targeting the mycobacterial 16S ribosomal RNA were synthesized. The cross-reactivity of MTB- and NTM-specific probes was examined with reference strains and 10 other frequently isolated bacterial species. A total of 140 sputum specimens were analyzed, comprising 100 MTB-positive specimens, 21 NTM-positive specimens, and 19 MTB/NTM-negative specimens; all of them were previously confirmed by PCR and culture. The PNA FISH test results were graded by using the United States Centers for Disease Control and Prevention-recommended scale and compared with the results from the fluorochrome acid-fast bacterial stain. RESULTS: The MTB- and NTM-specific PNA probes showed no cross-reactivity with other tested bacterial species. The test results demonstrated 82.9% agreement with the culture results with diagnostic sensitivity of 80.2% and diagnostic specificity of 100.0% (kappa=0.52, 95% confidence interval: 0.370-0.676). CONCLUSIONS: Dual-color PNA FISH showed high specificity for detecting and identifying mycobacteria in clinical specimens. However, because of its relatively low sensitivity, this method could be more applicable to culture confirmation. In application to direct specimens, the possibility of false-negative results needs to be considered.
Cell Wall ; Centers for Disease Control and Prevention (U.S.) ; DNA ; Fluorescence* ; In Situ Hybridization* ; Mycobacterium tuberculosis* ; Nucleic Acid Probes* ; Peptide Nucleic Acids ; Polymerase Chain Reaction ; RNA, Ribosomal, 16S ; Sputum

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

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

BACKGROUND: In Vietnam, we observed a high incidence of carbamazepine (CBZ)-induced severe cutaneous adverse drug reactions (SCARs)-Stevens-Johnson syndrome (SJS)/toxic epidermal necrolysis (TEN), and drug-induced hypersensitivity rash with eosinophilia and systemic symptoms (DRESS). In other Asian countries, HLA-B*1502 is an established risk factor for SCARs. OBJECTIVE: The aim of our study was to determine the frequency of HLA-B*1502 in SCARs patients at a large University Medical Center in Hanoi, Vietnam. METHODS: Thirty-eight cases of SCARs caused by CBZ and 25 patients with epilepsy tolerating CBZ were enrolled in a case-controlled study. Clinical manifestations and laboratory findings were recorded for each subject. Genomic DNA was isolated using the QIAamp DNA purification system. The combination of polymerase chain reaction and sequence specific oligonucleotide probes with the Luminex 100×MAP flow cytometry dual laser system was then used to quantitate fluorescently labelled oligonucleotides attached to colour-coded microbeads. RESULTS: Cases comprised 20 SJS (52.6%), 7 TEN (18.4%), 8 overlap syndrome (21.1%), and 3 DRESS patients (7.9%). A strong association between HLA B*1502 and bullous skin reactions such as SJS/TEN and overlap was confirmed with an odds ratio (OR) of 33.78 (95% confidence interval [CI], 7.55-151.03), p < 0.0001, Sensitivity 91.4%, Specificity 76.0%, positive predictive value 84.2%, and negative predictive value 86.4%. We did not, however, observe any correlation between the presence of this allele and CBZ-induced nonbullous skin reactions (DRESS) (OR, 6.33; 95% CI, 0.48-82.74; p = 0.1592). CONCLUSION: Our results indicate the presence of HLA-B*1502 in Vietnamese is a pharmacogenetic risk factor for developing CBZ-induced SJS/TEN.
Academic Medical Centers ; Alleles ; Asian Continental Ancestry Group ; Carbamazepine ; Case-Control Studies ; Cicatrix ; DNA ; Drug-Related Side Effects and Adverse Reactions ; Eosinophilia ; Epilepsy ; Exanthema ; Flow Cytometry ; HLA-B Antigens ; Humans ; Hypersensitivity ; Incidence ; Microspheres ; Odds Ratio ; Oligonucleotide Probes ; Oligonucleotides ; Pharmacogenetics ; Polymerase Chain Reaction ; Risk Factors ; Sensitivity and Specificity ; Skin ; Vietnam

The epidemiology on human papillomavirus (HPV) among human immunodeficiency virus (HIV)-infected women in Korea is not well established. A retrospective study was conducted to determine the prevalence and genotype distribution of HPV infection among HIV-infected women in Korea. HPV DNA genotype and cervical cytology were examined in 60 HIV-positive women and 1,938 HIV-negative women. HPV genotypes were analyzed by using a HPV DNA chip. HIV-infected women had higher prevalence of high-risk HPV (hr-HPV) infection (30% vs 4.9%, adjusted odds ratio [AOR], 6.96; 95% confidence interval [CI], 3.63-13.34, P<0.001) and abnormal cervical cytology (18.3% vs 1.8%, AOR, 10.94; 95% CI, 5.18-23.1, P<0.001) compared with controls. The most common hr-HPV genotype detected in HIV-infected women was HPV 16 (10%), followed by 18 (6.7%) and 52 (5%). Prevalence of quadrivalent vaccine-preventable types (HPV 6, 11, 16, and 18) was 21.7% and 2.3% in HIV-positive women and HIV-negative women, respectively. Age was a significant risk factor for hr-HPV infection in HIV-infected women (P=0.039). The presence of hr-HPV was significantly associated with abnormal cervical cytology (P<0.001). These findings suggest that HPV testing for cervical cancer screening in HIV-infected women would be necessary, particularly among young age group.
Adult ; Age Factors ; Cervix Uteri/virology ; DNA Probes, HPV/diagnostic use ; DNA, Viral/genetics ; Female ; Genotype ; HIV Infections/complications/*epidemiology/genetics ; HIV-1/genetics ; Human papillomavirus 11/genetics/isolation & purification ; Human papillomavirus 16/genetics/isolation & purification ; Human papillomavirus 18/genetics/isolation & purification ; Human papillomavirus 6/genetics/isolation & purification ; Humans ; Middle Aged ; Papillomavirus Infections/complications/*epidemiology/*genetics ; Prevalence ; Republic of Korea/epidemiology ; Retrospective Studies ; Risk Factors ; Uterine Cervical Neoplasms/epidemiology/genetics/virology

OBJECTIVETo compare the application values of real-time quantitative PCR-Sanger sequencing and TaqMan probe method in the detection of KRAS and BRAF mutations, and to correlate KRAS/BRAF mutations with the clinicopathological characteristics in colorectal carcinomas.

METHODSGenomic DNA of the tumor cells was extracted from formalin fixed paraffin embedded (FFPE) tissue samples of 344 colorectal carcinomas by microdissection. Real-time quantitative PCR-Sanger sequencing and TaqMan probe method were performed to detect the KRAS/BRAF mutations. The frequency and types of KRAS/BRAF mutations, clinicopathological characteristics and survival time were analyzed.

RESULTSKRAS mutations were detected in 39.8% (137/344) and 38.7% (133/344) of 344 colorectal carcinomas by using real-time quantitative PCR-Sanger sequencing and TaqMan probe method, respectively. BRAF mutation was detected in 4.7% (16/344) and 4.1% (14/344), respectively. There was no significant correlation between the two methods. The frequency of the KRAS mutation in female was higher than that in male (P < 0.05). The frequency of the BRAF mutation in colon was higher than that in rectum. The frequency of the BRAF mutation in stage III-IV cases was higher than that in stageI-II cases. The frequency of the BRAF mutation in signet ring cell carcinoma was higher than that in mucinous carcinoma and nonspecific adenocarcinoma had the lowest mutation rate. The frequency of the BRAF mutation in grade III cases was higher than that in grade II cases (P < 0.05). The overall concordance for the two methods of KRAS/BRAF mutation detection was 98.8% (kappa = 0.976). There was statistic significance between BRAF and KRAS mutations for the survival time of colorectal carcinomas (P = 0.039). There were no statistic significance between BRAF mutation type and BRAF/KRAS wild type (P = 0.058).

CONCLUSIONS(1) Compared with real-time quantitative PCR-Sanger sequencing, TaqMan probe method is better with regard to handling time, efficiency, repeatability, cost and equipment. (2) The frequency of the KRAS mutation is correlated with gender. BRAF mutation is correlated with primary tumor site, TNM stage, histological types and histological grades.(3) BRAF gene mutation is an independent prognostic marker for colorectal carcinomas.

Adenocarcinoma ; genetics ; pathology ; Adenocarcinoma, Mucinous ; genetics ; pathology ; Adult ; Aged ; Aged, 80 and over ; Carcinoma, Signet Ring Cell ; genetics ; pathology ; Colonic Neoplasms ; genetics ; pathology ; Colorectal Neoplasms ; genetics ; pathology ; DNA Mutational Analysis ; Female ; Humans ; Male ; Middle Aged ; Mutation ; Neoplasm Grading ; Neoplasm Staging ; Oligonucleotide Probes ; Proto-Oncogene Proteins ; genetics ; Proto-Oncogene Proteins B-raf ; genetics ; Proto-Oncogene Proteins p21(ras) ; Real-Time Polymerase Chain Reaction ; Rectal Neoplasms ; genetics ; pathology ; Sequence Analysis, DNA ; Sex Factors ; Young Adult ; ras Proteins ; genetics