The aim of this study was to investigate the factors which affect HLA typing in 311 umbilical cord blood (UCB) samples. The HLA low resolution typing of UCB samples with misinterpreted HLA types from 311 UCB samples analyzed by PCR-SSO and PCR-SSP was performed. 7 samples difficult to determine their HLA genotype were sequenced directly and the reason leading to misinterpret HLA typing was analyzed. The results indicated that 99.4% of misinterpreted samples resulted from the restriction of HLA typing method itself and 0.6% of misinterpreted samples were suspected to be contaminated with maternal blood in UCB. It is concluded that HLA typing is mainly affected by the shortcomings of oligonucleotide probe design for PCR-SSO and lack of allele specific primers of PCR-SSP.
Alleles ; Base Sequence ; DNA Primers ; Fetal Blood ; immunology ; Genotype ; HLA Antigens ; genetics ; Histocompatibility Testing ; methods ; Humans ; Oligonucleotide Probes ; Polymerase Chain Reaction ; methods

Over a long period of time, studies on HLA structure and function have been the research hotspots. for it is very important to understand the essential of life science and disease mechanism. With the rapid development of molecular biology, HLA typing makes great progress. It has changed from traditional serological typing to DNA-based typing. More and more HLA genotyping methods have been developed and applied. In this essay, the author reviewed and appraised all kinds of HLA genotyping techniques and introduced two new techniques.
DNA Primers ; Genetic Techniques ; Genotype ; HLA Antigens/genetics* ; Histocompatibility Testing/methods* ; Humans ; Oligonucleotide Array Sequence Analysis ; Oligonucleotide Probes ; Polymerase Chain Reaction/methods* ; Polymorphism, Restriction Fragment Length ; Polymorphism, Single-Stranded Conformational ; Sequence Analysis, DNA/methods*

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

The amplification of c-myc oncogene was evaluated in 42 cases of ovarian carcinomas to correlate with clinical parameters. Using oligonucleotide primers, sequences from the c-myc exon-3 gene and from a control gene, tissue plasminogen activator (tPA), were amplified simultaneously by polymerase chain reaction (PCR). After the products of differential PCR (d-PCR) were electrophoresed, slot blot hybridization was performed, and hybridized with P32 dATP-labeled myc and tPA oligonucleotide probes and then autoradiographed. The signal intensities of the two products were quantitated by densitometry and the ratios of two products (c-myc/tPA) were measured. The ovarian carcinomas showed significantly increased amplification of c-myc oncogene Oligonucleoti compared to normal control group (p<0.05). 15 of 42 cases (35.7%) showed various degrees of the MYC gene amplification up to 27 folds in various histologic types of ovarian carcinomas. No significant differences of the MYC gene amplification according to histologic subtypes, tumor action) grades and clinical stages of ovarian carcinomas were present.
Densitometry ; DNA Primers ; Genes, myc ; Oligonucleotide Probes ; Oncogenes* ; Polymerase Chain Reaction* ; Tissue Plasminogen Activator

Cervical Intraepithelial Neoplasia ; diagnosis ; pathology ; virology ; China ; DNA Probes, HPV ; Female ; Humans ; Pregnancy ; Vaginal Smears ; methods

OBJECTIVETo develop a real-time polymerase chain reaction(PCR) based on TaqMan technology by using a new MGB probe for detecting enterotoxigenic Escherichia coli (ETEC) in paper.

METHODSPrimers and MGB probe were designed in the ecoding region of heat-stable toxin of ETEC. Real-time PCR detected ETEC by using the exterior standard method with protracting standard curves. The specificity, sensitivity, accuracy, stability of real-time PCR system was evaluated. An internal negative antithesis was added to the real-time PCR system in order to get rid of the false positive of system. Using UNG enzyme expelled the contamination of PCR reaction.

RESULTSPrimers and MGB probe were suited to the Real-time PCR. The assay showed that the method was quick, special, sensitive and stable. The real-time PCR system could detect ETEC in a large scale. The assay might be finished in two hour.

CONCLUSIONThese observations suggested that real-time PCR based on MGB probe should be an excellent candidate for a standard ETEC detection method.

Bacterial Toxins ; isolation & purification ; DNA Primers ; DNA Probes ; DNA, Bacterial ; Enterotoxigenic Escherichia coli ; isolation & purification ; Molecular Probe Techniques ; Polymerase Chain Reaction ; methods ; Taq Polymerase

OBJECTIVETo develop a TaqMan real-time PCR for the detection of Aeromonas hydrophila.

METHODSThe conserved region of major adhesion gene of Aeromonas hydrophila (aha) was used to design primers and TaqMan probe. A total of six concentration gradients for forward and reverse primers ranging from 200 -700 nmol/L were chosen, and four concentration gradients for probe ranging from 100 - 400 nmol/L were chosen. And then the concentration of primers and probe were optimized by ANOVA of completely randomized design respectively. The specificity of the established method was evaluated by using bacteria as contrast, including 45 strains Vibrio cholerae, 20 strains Vibrio parahaemolyticus, 10 strains Vibrio fluvialis, 4 strains Vibrio mimicus, 5 strains Vibrio vulnificus, 1 strain Vibrio alginolyticus, 1 strain Vibrio furnissii, 5 strains Salmonella, 10 strains Shigella and 2 strains Plesiomonas shigelloides. The sensitivity, bacterial sensitivity and DNA sensitivity included,were evaluated. The stool of healthy people was contaminated by Aeromonas hydrophila artificially, and the ability of the established TaqMan real-time PCR system for detection of Aeromonas hydrophila was also evaluated.

RESULTSThe cycle threshold (Ct) value deserved from 6 groups of primers concentration gradient was (x +/- s):20.69 +/- 0.33, 20.72 +/- 0.21, 20.81 +/- 0.12, 20.74 +/- 0.12, 20.51 +/- 0.16 and 20.69 +/- 0.11, respectively, and the concentration of forward primer and reverse primer was determined to be 200 nmol/L (F = 1.33, P = 0.28). The Ct value deserved from 4 groups of probe concentration gradient was (x +/- s):20.56 +/- 0.08, 20.82 +/- 0.05, 20.82 +/- 0.11 and 20.93 +/- 0.09, respectively, and the concentration of probe was determined to be 100 nmol/L (F = 5.26, P = 0.01). The bacterial sensitivity and DNA sensitivity were 80 CFU/ml and 100 fg/microl respectively, and the sensitivity to detect Aeromonas hydrophila from stool was 8 x 10(3) CFU/ml, which might be 8 CFU/ml after 8 hours' enrichment. No amplification was observed in the templates of other bacterial.

CONCLUSIONThe TaqMan real-time PCR method targeting the aha gene of Aeromonas hydrophila had a high sensitivity and specificity and might be used to detect Aeromonas hydrophila from pure bacterial and stool rapidly.

Aeromonas hydrophila ; genetics ; DNA Primers ; Genes, Bacterial ; Molecular Probes ; Polymerase Chain Reaction ; methods ; Sensitivity and Specificity ; Species Specificity

BACKGROUND: Self-collection of secretion samples for HPV testing is a feasible alternative method for women who would decline to participate in population based cervical cancer programs. The purpose of this study was to determine the sensitivity and specificity of self-sampling for HPV in determining high grade squamous intraepithelial lesion (HSIL) using the pad, and we also wished to compare the results from samples collected by women themselves and those results from samples collected by physicians. METHODS: Fifty patients voluntarily participated in the sensitivity and specificity study at the university hospitals and 290 volunteers participated in the agreement study at local clinics. DNA was extracted and amplified using HPV L1 consensus primers for the direct sequencing of the pad samples. RESULTS: For the detection of HSIL, self-collected pad sampling showed good sensitivity (75.0%) and excellent specificity (100%). Two hundreds eighty-six samples from the pads and concurrent physicians?samples showed the agreement at 98.6% with the Kappa, 0.9622 (p=0.0000). CONCLUSIONS: A self-sampling method using the pad for the detection of HPV DNA is suggested to be an efficient method to access many women for screening easily, rapidly and conveniently. Testing the pad method? utility for a country- or large area-based mass screening study will be necessary in the future.
Consensus ; DNA ; DNA Probes, HPV ; Female ; Hospitals, University ; Humans ; Mass Screening ; Sensitivity and Specificity ; Uterine Cervical Neoplasms ; Volunteers

OBJECTIVETo identify HLA novel allele in Chinese Han individual.

METHODSAn unknown HLA-B allele which was similar to HLA-B*5610 was detected by polymerase chain reaction-sequence specific oligonucleotide probes(PCR-SSOP), PCR-sequence specific primer(PCR-SSP) and heterozygous sequence-based typing (SBT) in a Chinese Han individual. Its anomalous patterns suggested the possible presence of new allele. The HLA-B*56 allele was amplified separately by using allele-specific primers and sequencing exons 2-4 in both directions. The differences between the novel B*56 allele and B 5610 were identified.

RESULTSThere were 4nt changes from B*5610 in exon 3, at nt379 where C>G (codon 127 CTG>GTG, 127 Leu>Val); nt412 where A>G (codon 138 AAC>GAC, 138 Asn>Asp), nt419 where T>C and nt420 where A>C (codon 140 TTA>TCC, 140 Leu>Ser). The sequence was submitted to Genbank and the accession number was EF016753.

CONCLUSIONThis allele is a novel HLA-B allele, and has been officially named HLA-B*5618 by the WHO Nomenclature Committee in September 2006.

Alleles ; China ; ethnology ; Ethnic Groups ; genetics ; Exons ; Gene Frequency ; HLA-B Antigens ; genetics ; Haplotypes ; Heterozygote ; Humans ; Male ; Oligonucleotide Probes ; genetics ; Polymerase Chain Reaction ; Sequence Analysis, DNA

OBJECTIVETo develop a technique for simultaneous detection of various target genes in Roundup Ready soybean by combining multiplex PCR and low-density DNA microarray.

METHODSTwo sets of the multiplex PCR system were used to amplify the target genes in genetically modified (GM) soybean. Seventeen capture probes (PCR products) and 17 pairs of corresponding primers were designed according to the genetic characteristics of Rroundup Ready soybean (GTS40-3-2), maize (Mon810, Nk603, GA21), canola (T45, MS1/RF1), and rice (SCK) in many identified GM crops. All of the probes were categorized and identified as species-specific probes. One negative probe and one positive control probe were used to assess the efficiency of all reactions, and therefore eliminate any false positive and negative results. After multiplex PCR reaction, amplicons were adulterated with Cy5-dUTP and hybridized with DNA microarray. The array was then scanned to display the specific hybridization signals of target genes. The assay was applied to the analysis of sample of certified transgenic soybean (Roundup Ready GTS40-3-2) and canola (MS1/RF1).

RESULTSA combination technique of multiplex PCR and DNA microarray was successfully developed to identify multi-target genes in Roundup Ready soybean and MS1/RF1 canola with a great specificity and reliability. Reliable identification of genetic characteristics of Roundup Ready of GM soybean from genetically modified crops was achieved at 0.5% transgenic events, indicating a high sensitivity.

CONCLUSIONA combination technique of multiplex PCR and low-density DNA microarray can reliably detect and identify the genetically modified crops.

Base Sequence ; Cloning, Molecular ; Crops, Agricultural ; DNA Primers ; DNA Probes ; Oligonucleotide Array Sequence Analysis ; Plants, Genetically Modified ; Polymerase Chain Reaction ; methods