Improvement of sensitivity in HLA-DRB1 typing by semi-nested PCR-RFLP

A sensitive method of HLA-DRB1 typing was devised using a semi-nested polymerase chain reaction (PCR) followed by a restriction fragment length polymorphism (RFLP) analysis (semi-nested PCR-RFLP method). The first-round amplification (30 cycles) of the semi-nested PCR was performed using DRB generic primer pairs and the second round of PCRs (20 cycles) were performed using DRB1 group-specific primers. The products of the second round PCRs were digested with restriction endonucleases for the typing of HLA-DRB1 alleles. By this method, HLA-DRB1 typing was possible from 10 pg of genomic DNA extracted from lymphocytes and from 0.5 microliter of 1,000 times diluted blood without DNA extraction. HLA-DRB1 alleles could be typed from a 2-mm long bloodstained cotton thread prepared from 10 times diluted blood and from a 2-mm thread of whole blood bloodstains stored at room temperature for 2 years. From the mixture of blood of two individuals with different genotypes, DRB1 alleles of the minor component were detected down to 1/1,000 of the major component. This semi-nested PCR-RFLP method is useful for HLA-DRB1 typing from extremely small amounts of DNA and from mixed samples.     

HLA-DOB1 "low-resolution' typing by PCR amplification with sequence-specific primers (PCR-SSP)

In the study described here primers were designed for DQB1 'low-resolution', i.e. generic, typing by PCR amplification with sequence-specific primers (PCR-SSP) considering all the currently recognized DQB1 alleles, i.e. 0501-0504, 0601-0609, 0201, 0301-0305 and 0401-0402. This resolution was achieved by performing eight PCR reactions per individual. The DQB1 alleles corresponding to the serological specificities DQ4, DQ5, and DQ6 were uniquely identified, whereas the DQ2, DQ7, DQ8 and DQ9 specificities were amplified by two primer mixes. All homozygous and heterozygous combinations of the serological series DQ1 to DQ9 could be distinguished. The yield of amplified products were increased compared to our previously described DQB1 'high-resolution' typing technique by lengthening many of the primers, modifying the PCR cycling parameters and by including glycerol in the PCR reaction mixtures. Thirty-one cell lines and 90 donor spleen cells were investigated by the DQB1 'low-resolution' PCR-SSP technique as well as by TaqI DRB-DQA-DQB RFLP analysis. The concordance between PCR-SSP typing and RFLP analysis was 100%. The cell lines and 20 of the spleen cells were typed twice with complete reproducibility. No false positive or false negative typing results were obtained. DQB1 'low-resolution' PCR-SSP typing, including DNA extraction, PCR amplification, gel detection, documentation and interpretation, were performed in 2 h which renders the PCR-SSP technique suitable also for the genotyping of cadaveric organ donors.     

Numbers and ratios of X-chromosomal-linked opsin genes

Quantitative Southern blotting and PCR/RFLP analysis were used to determine the number and ratio of long-wave-sensitive (L-) and mid-wave-sensitive (M-) opsin genes in 25 colour-normal caucasian males. The average observed ratio was 1:2.8 +/- 1.2 for Southern blot analysis and 1:3.0 +/- 1.7 for PCR/RFLP analysis. Thus, the two techniques yielded similar results for the ratio of L- to M-opsin genes (Wilcoxon t-test, P < 0.01). PCR/RFLP analysis of a Sma I polymorphism specific for the most proximal opsin gene suggested an average gene number of 6.0 +/- 2.1, with a range from 4 to 12 in individual subjects. In contrast, Southern blot analysis suggested an average number of 3.8 +/- 1.2, with a range from 2 to 7 (on the assumption that only one L-opsin gene is ever present). Differences between the L- to M-opsin gene ratio and the total gene number in some subjects may result from the presence of multiple L-opsin genes and/or hybrid opsin genes in colour-normal males. An exact determination of the total gene number will require employing other molecular techniques.     

A T1083C polymorphism in the human adenosine A2a receptor gene is not associated with essential hypertension

The adenosine A2a receptor (A2aAR) gene is thought to be involved in essential hypertension because adenosine elicits vasodilation and decreases arterial blood pressure via this receptor, and because disruption of the A2aAR gene increases blood pressure in mice. Therefore, using a restriction fragment length polymorphism (RFLP) of the A2aAR gene, we performed an association study in patients with essential hypertension. One hundred forty-two patients with essential hypertension and 142 age-matched subjects with normal blood pressure were studied. Polymerase chain reaction (PCR) was applied to amplify the T1083C polymorphic site in the A2aAR gene, and restriction analysis of the PCR product was employed to score the T and C alleles. Overall distributions of allele frequencies in the two groups were not significantly different. Thus, the alleles detected by this RFLP polymorphism in the A2aAR gene are not associated with essential hypertension.     

Improved amplification of microbial DNA from blood cultures by removal of the PCR inhibitor sodium polyanetholesulfonate

Molecular methods are increasingly used to identify microbes in clinical samples. A common technical problem with PCR is failed amplification due to the presence of PCR inhibitors. Initial attempts at amplification of the bacterial 16S rRNA gene from inoculated blood culture media failed for this reason. The inhibitor persisted, despite numerous attempts to purify the DNA, and was identified as sodium polyanetholesulfonate (SPS), a common additive to blood culture media. Like DNA, SPS is a high-molecular-weight polyanion that is soluble in water but insoluble in alcohol. Accordingly, SPS tends to copurify with DNA. An extraction method was designed for purification of DNA from blood culture media and removal of SPS. Blood culture media containing human blood and spiked with Escherichia coli was subjected to an organic extraction procedure with benzyl alcohol, and removal of SPS was documented spectrophotometrically. Successful amplification of the extracted E. coli 16S rRNA gene was achieved by adding 5 microliter of undiluted processed sample DNA to a 50-microliter PCR mixture. When using other purification methods, the inhibitory effect of SPS could be overcome only by dilution of these samples. By our extraction technique, even uninoculated blood culture media were found to contain bacterial DNA when they were subjected to broad-range 16S rRNA gene consensus PCR. We conclude that the blood culture additive SPS is a potent inhibitor of PCR, is resistant to removal by traditional DNA purification methods, but can be removed by a benzyl alcohol extraction protocol that results in improved PCR performance.     

Molecular fingerprinting of mycobacterium tuberculosis isolates obtained in havana, cuba, by IS6110 restriction fragment length polymorphism analysis and by the double-repetitive-element PCR method

Mycobacterium tuberculosis sputum isolates from 38 patients, obtained in the first 6 months of 1997 in Havana, Cuba, were characterized by IS6110 restriction fragment length polymorphism (RFLP) analysis and the double-repetitive-element PCR (DRE-PCR) method. Among 41 strains from 38 patients, 24 and 25 unique patterns, and 5 and 4 cluster patterns, were found by the RFLP and DRE-PCR methods, respectively. Patients within two of these clusters were found to be epidemiologically related, while no relation was observed in patients in the other clusters. The DRE-PCR method is rapid, and it was as discriminating as IS6110 RFLP analysis in identifying an epidemiological association. Its simplicity makes the technique accessible for subtyping of M. tuberculosis strains in laboratories not equipped to perform RFLP analysis.     

Genotypic selection methods for the direct analysis of point mutations

Genotypic selection enriches a particular DNA sequence relative to another closely-related DNA sequence based only on a change of one or a few bases. This review is a survey of the genotypic selection methods that have the sensitivity to detect rare point mutations. These methods are primarily being used to study mutations caused by environmental mutagens; however, the ability to detect and measure very minor DNA sequence populations is likely to further research efforts in many fields. The approaches for allele-selection have intrinsic strengths and weaknesses, and vary greatly in sensitivity. The most sensitive method is Restriction Fragment Length Polymorphism/Polymerase Chain Reaction (RFLP/PCR) by which mutant fractions as low as 1 mutant allele in 10(8) wild-type alleles can be detected. The RFLP/PCR approach is presented as a prototype genotypic selection method. Genotypic selection methods are categorized in terms of those that (1) selectively destroy the abundant or wild-type allele, (2) selectively amplify the rare or mutant allele, or (3) spatially separate the alleles. Issues relevant to the further development of genotypic selection methods include initial DNA pool size, strategies to eliminate the bulk of extraneous DNA, the use of an internal copy number standard in quantitative PCR, the fidelity of thermostable DNA polymerases, and the effective use of PCR in linking two or more genotypic selection techniques. We conclude that proficient genotypic selection requires more than one allele-enrichment technique with at least one of these preceding a high-fidelity PCR amplification step.     

Genetic diversity of Borrelia burgdorferi in lyme disease patients as determined by culture versus direct PCR with clinical specimens

Two hundred seventeen isolates of Borrelia burgdorferi originally cultured from skin biopsy samples or blood of early Lyme disease patients were genetically characterized by PCR-restriction fragment length polymorphism (RFLP) typing of the 16S-23S ribosomal DNA intergenic spacer. Three major RFLP types were observed. Of the cultured isolates, 63 of 217 (29.0%) were type 1, 85 of 217 (39.2%) were type 2, and 58 of 217 (26.7%) were type 3; mixtures of two RFLP types were obtained in 6.0% (13 of 217) of the cultures. Comparison of typing of B. burgdorferi performed directly on 51 patient skin specimens with typing of cultures originally isolated from the same tissue revealed that a much larger proportion of direct tissue samples had mixtures of RFLP types (43.1% by direct typing versus 5.9% by culture [P < 0.001). In addition, identical RFLP types were observed in only 35.5% (11 of 31) of the paired samples. RFLP type 3 organisms were recovered from blood at a significantly lower rate than were either type 1 or type 2 strains. These studies demonstrate that the genetic diversity of B. burgdorferi patient isolates as determined by cultivation differs from that assessed by PCR performed directly on patient tissue.     

A molecular epidemiologic investigation of north Asia fever in scenic spots of Beijing suburb

PCR/RFLP technique was used to detect spotted fever group rickettsiae (SFGR) in ticks and small mammals collected in eleven scenic spots of Beijing suburb. We not only detected Rickettsia sibirica in D. sinicus and hedgehog collected nearby the Museum of Aviation, but also isolated two strains of SFGR from them, named as BJ-95 strain and BJH-95 strain respectively. The two strains were identified as R. sibirica by SDS-PAGE, Western blot and PCR/RFLP. The results demonstrated the existence of horizontal transmission of R. sibirica between ticks and small mammals and showed the most scenic spots except the vicinity of Museum of Aviation being investigated were safe to North Asia Fever. This is the first report on the isolation of R. sibirica in hedgehogs.     

PCR in combination with silver as a method for the determination of apolipoprotein E alleles

OBJECTIVE: To present a rapid, sensitive and safe procedure for the determination of human apolipoprotein E alleles. METHODS: Genomic DNA was extracted from 0.5ml whole blood by a non-phenol protocol. After PCR and restriction enzyme digestion,short DNA fragments were detected by a simplified silver staining method. This technique was used to assay apolipoprotein E alleles in 19 patients with sporadic Alzheimer's disease and 41 normal aged persons. RESULTS: The short DNA fragments were visualized clearly on polyacrylamide gel processed by silver staining. It took 24 hours from DNA extraction to the final result. The A4 allele frequency in patients with sporadic Alzheimer's disease was 0.29, much higher than that in control group (0.02, P<0.001). CONCLUSION: PCR in combination with silver staining can satisfy the need for high-resolution and high-efficiency in the determination of apolipoprotein E alleles and can be used as a routine procedure in clinical and epidemiological investigations.     

Detection and characterization of the fimA gene of Escherichia coli O157:H7

An expected 850-bp DNA fragment containing fimA, the structural gene for type 1 fimbriae, and flanking sequences was amplified from 39 (of 46) pathogenic and commensal strains of Escherichia coli using the polymerase chain reaction (PCR). Restriction fragment length polymorphism (RFLP) analysis of the amplified products showed 13 HinP1 and four Sau961 restriction profiles among these 39 E. coli strains, revealing the polymorphic nature of this allele. A unique RFLP pattern was shared by E. coli O157:H7, O157:H- and a few O55 serotype strains. DNA sequence analysis of the fimA region demonstrated that E. coli O157:H7 strain 933 and O157:H- strain E32511 contained identical DNA sequences that were distinct from other E. coli strains, especially a 16-bp sequence 5' to fimA that was conspicuously absent only in E. coli O157 strains. Exploiting these differences, a PCR assay was developed that amplifies a 936-bp fragment from all E. coli O157:H7 strains examined to date. This PCR assay offers a simple, rapid, and reliable means to detect E. coli strains of the O157:H7 serotype.     

Effect of aspirin in coronary artery bypass grafting

OBJECTIVES: To evaluate the effect of aspirin (ASA) therapy on postoperative blood loss, transfusion requirements, reoperation for bleeding, duration of stay in the intensive care unit and in the hospital in a selected population undergoing a first coronary artery bypass grafting (CABG) surgery. DESIGN: Prospective observational study in consecutive patients during a 3-month period. SETTING: A teaching cardiothoracic center. PARTICIPANTS: Two hundred forty consecutive patients undergoing elective coronary artery bypass grafting surgery for the first time. INTERVENTIONS: Two hundred forty consecutive patients admitted for a first CABG the day before surgery were visited. patients with an abnormal routine coagulation screen or taking drugs that might have affected their coagulation mechanisms were prospectively excluded (n = 96). The date of the last dose of ASA was recorded in the 144 remaining patients, and data were acquired prospectively. MEASUREMENTS AND MAIN RESULTS: Total mediastinal blood drainage, blood products usage, reopening, and duration of intensive care unit and hospital stay were recorded. Patients were grouped by days free of ASA. There were no significant differences detected between groups. CONCLUSIONS: In patients undergoing a first CABG and with no known factors affecting their coagulation, ASA therapy did not appear to increase blood loss, reopening for bleeding, or blood products usage requirements during the hospital stay. ASA therapy did not influence the duration of stay in intensive care or in the hospital.     

Clinical interview skills and identification of emotional disorders in primary care

BACKGROUND: This study focuses on the detection of medically compromised dental patients in the Netherlands by means of a validated patient-administered medical risk-related history (MRRH). Due to social changes and scientific innovations in the past decade, more medically compromised patients will be needing special dental treatment. METHODS: The medical problems of 29,424 dental patients (age 18 years and over) from 50 dental practices in the Netherlands were registered by means of the MRRH. The patients were classified according to the ASA risk-score system, which was modified for dental treatment. An inventory of the number and nature of medical problems and the modified ASA risk score was drawn up in relation to dental treatment and age. RESULTS: The average age of the patients was 37.1 +/- 13.5 years. According to the current guidelines, dental treatment must be modified if the patient has an ASA score of III or IV. A relatively high percentage of patients ages 65-74 (23.9%) and 75 or over (34.9%) did have an ASA score of III or IV. Furthermore, the medical problems were classified into 10 categories, and the relationship to age was examined. The conditions that increased with age were hypertension and cardiovascular, neurological, endocrinological, infectious, and blood diseases. CONCLUSIONS: For the dental practice, these results mean that the MRRH can play an important role in adapting dental treatment to the specific needs of patients. This is especially important in the case of elderly patients.     

Taste disks are induced in the lingual epithelium of salamanders during metamorphosis

We have established a practical method of complete high-resolution typing for all HLA-A alleles using the polymerase chain reaction (PCR)-restriction fragment-length polymorphism (RFLP) technique combined with allele group- and sequence-specific amplification. The second and third exons of the HLA-A gene, in which most allelic variations are observed, were separately amplified by PCRs with 3 and 4 group-specific primer pairs, respectively. Each PCR-amplified product was digested by allele-specific restriction endonucleases and then subjected to electrophoresis on a 10% polyacrylamide gel. In this way, 62 out of 79 HLA-A alleles could be discriminated by the RFLP patterns derived from the genetic polymorphism in the exon 2 and 3 domains. The remaining 17 alleles could be defined unequivocally by either PCR-RFLP analysis after exon 4 amplification or PCR analysis with sequence-specific primers (SSP). By this method, complete HELA-A genotyping for all homozygous and heterozygous combinations can be accomplished, establishing technically simple, economical and practical routine typing of the HLA-A gene, especially for small samples.     

Atopic keratoconjunctivitis

Rapid, non-culture, serogroup determination of meningococcal infection is important in contact management where vaccination may be possible. The impending availability of polysaccharide-protein conjugate vaccines for serogroup C disease requires maximal case ascertainment, with serogroup determination, at a time when the number of culture confirmed meningococcal infections is decreasing. A polymerase chain reaction assay (PCR), based on a restriction fragment length polymorphism (RFLP) in the meningococcal serogroup B and C sialytransferase (siaD) gene, was developed to combine the non-culture diagnosis of meningococcal infection from CSF, whole blood and serum with serogroup (B and C) identification. The PCR assay was adapted to an ELISA format incorporating hybridization with serogroup-specific B and C oligonucleotide probes. Specificity for CSFs was 100% and sensitivities were respectively 81, 63 and 30% for CSFs, whole blood and sera. The serogroup-specific PCR ELISA is a significant addition to currently available tests for non-culture diagnosis of meningococcal infection and outbreak investigation.     

Inhibition of platelet-vessel wall interactions by thromboxane receptor antagonism in a human in vitro system: potentiation of antiplatelet effects of aspirin

BACKGROUND: Pharmacological inhibition of arachidonic acid metabolism has proven therapeutically useful in the prevention of cardiovascular events. METHODS: We have investigated the ability of Bay u 3405, a synthetic thromboxane antagonist, to interfere with platelet aggregation and arachidonic acid metabolism. The antiplatelet action was also analysed in a perfusion system in which vascular subendothelium was exposed to circulating human blood (10 min; shear rate = 800 s-1). Platelet interactions were morphometrically analysed and results compared with those obtained in studies with blood from donors taking aspirin (acetylsalicylic acid, ASA) (500 mg day-1). The additional effect of Bay u 3405 on the antiplatelet action of ASA was also evaluated. RESULTS: Bay u 3405 caused a dose-dependent inhibition of platelet aggregation induced by U46619 with a maximal effect at concentrations > or = 0.01 microgram mL-1. Higher concentrations (> or = 0.05 micrograms mL-1) also inhibited aggregations induced by ADP or collagen. Bay u 3405 did not interfere with platelet arachidonic acid metabolism. In perfusion studies, Bay u 3405 (0.01 microgram mL-1) significantly decreased the total surface of the vessel covered by platelets (%CS = 18.7 +/- 1.09 vs. 24.4 +/- 1.94; P < 0.05) and the formation of large aggregates %T = 7.5 +/- 0.87 vs. 19.3 +/- 1.61; P < 0.01). ASA treatment reduced platelet aggregate formation (%T = 13.7 +/- 2.06; P < 0.05) but did not affect the total surface covered by platelets. The in vitro addition of Bay u 3405 to blood from ASA-treated donors further reduced the formation of large aggregates (%T = 2.7 +/- 0.79; P < 0.01 vs. ASA). CONCLUSIONS: In vitro effect of Bay u 3405 on platelet function were superior to those observed with ASA. The thromboxane antagonism antagonism provided by Bay u 3405 further enhanced the inhibition of platelet aggregate formation found after ASA treatment.     

Molecular typing of Borrelia burgdorferi from Lyme disease patients by PCR-restriction fragment length polymorphism analysis

Ninety-three Borrelia burgdorferi isolates obtained from erythema migrans lesions or blood of Lyme disease patients in Westchester County, N.Y., between 1991 and 1994 were characterized by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the 16S-23S rRNA gene spacer. All isolates could be classified into three distinct RFLP types. Among the 82 skin biopsy isolates studied, 21 (25.6%) were type 1, 37 (45.1%) were type 2, and 21 (25.6%) were type 3. Three (3.7%) cultures contained a mixture of two isolates with distinct RFLP types. The 11 isolates cultured from blood showed a similar predominance of RFLP type 2 (6 of 11; 54.5%) relative to types 1 (2 of 11; 18.2%) and 3 (3 of 11; 27.3%). For one patient both skin and blood isolates were cultured, and RFLP analysis revealed that these isolates differed from one another. This study demonstrates that there is genotypic heterogeneity in B. burgdorferi strains infecting Lyme disease patients, and this typing approach may allow differentiation of isolates with various degrees of pathogenic potential.     

Comparative study of five methods for intratypic differentiation of polioviruses

A coded panel of 90 poliovirus isolates, 30 of each of the three known serotypes, was used to evaluate five methods for the intratypic differentiation of polioviruses: (i) an enzyme-linked immunosorbent assay with polyclonal cross-absorbed antisera (PAb-E), (ii) a neutralization assay with type-specific monoclonal antibodies (MAb-N), (iii) a restriction fragment length polymorphism (RFLP) assay, (iv) a Sabin vaccine strain-specific PCR assay, and (v) a Sabin vaccine strain-specific cRNA probe hybridization (ProHyb) assay. Sequence analysis was used for the definitive characterization of the strains. The panel was distributed to five laboratories; each laboratory analyzed the strains by at least two methods. Each method was used by three or four laboratories. The total performance scores (percentage correct results per number of tests) of the five methods were 96.7% for PAb-E, 93.9% for MAb-N, 91.9% for RFLP assay, 93.3% for Sabin vaccine strain-specific PCR, and 97.4% for Sabin vaccine strain-specific ProHyb. Consistent results were obtained by each laboratory for 88 of 90 isolates (97.8%) examined by PAb-E, 81 of 90 isolates (90.0%) examined by MAb-N, 78 of 90 isolates (86.7%) examined by RFLP assay, 81 of 90 isolates (90.0%) examined by PCR, and 89 of 90 isolates (98.9%) examined by ProHyb assay. Six strains were classified differently by different methods. It is recommended that at least two methods be used for the intratypic differentiation of poliovirus isolates, and each method should be based on a different principle (i.e., antigenic properties and nucleotide sequence composition). If two assays yield discrepant results, further characterization, preferably by partial sequence determination, will be required for correct identification.     

Development of a polymerase chain reaction/restriction fragment length polymorphism method for Saccharomyces cerevisiae and Saccharomyces bayanus identification in enology

Several yeast strains of the species Saccharomyces cerevisiae, S. bayanus and S. paradoxus, first identified by hybridization experiments and measurements of DNA/DNA homology, were characterized using polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analysis of the MET2 gene. There was no exception to the agreement between this method and classical genetic analyses for any of the strains examined, so PCR/RFLP of the MET2 gene is a reliable and fast technique for delimiting S. cerevisiae and S. bayanus. Enological strains classified as S. bayanus, S. chevalieri, and S. capensis gave S. cerevisiae restriction patterns, whereas most S. uvarum strains belong to S. bayanus. Enologists should no longer use the name of S. bayanus for S. cerevisiae Gal strains, and should consider S. bayanus as a distinct species.     

Adenocarcinoma of the pancreas: detection of occult metastases in regional lymph nodes by a polymerase chain reaction-based assay

BACKGROUND: Stage I (T1-2NOM0) adenocarcinoma of the pancreas is associated with a 5-year survival rate of 15-25%. Despite apparently curative resection and pathologic staging indicating localized disease, these cancers recur. The authors hypothesized that there exists microscopic regional disease that is not detected by surgical exploration or routine histopathology. METHODS: Because 90-95% of pancreatic cancers exhibit codon 12 K-ras mutations, the authors examined regional lymph nodes for mutated K-ras as a marker of metastasis. DNA was extracted from paraffin embedded archival specimens (primary tumors and histologically negative lymph nodes) of patients with Stage I pancreatic adenocarcinoma. The target region of K-ras was amplified by polymerase chain reaction (PCR) and tested for codon 12 mutation by BstN1 restriction digestion (restriction fragment length polymorphism [RFLP]) that recognized normal but not mutated sequences. Cell lines that harbored normal or mutated K-ras and resected jejunum or gallbladder were used as controls. The regional lymph nodes of 22 patients whose tumors harbored mutated K-ras were tested. RESULTS: Dilution experiments with normal and mutant control cell line DNA demonstrated an assay sensitivity for mutated K-ras of 0.1%. Mutated K-ras was found in at least 1 regional lymph node in 16 (73%) of 22 patients with pathologic Stage I pancreatic adenocarcinoma, which suggested metastases not detected by routine histopathology. DNA sequence analysis was performed in four patients and confirmed identical point mutations in the primary tumor and accompanying PCR/RFLP positive lymph nodes. CONCLUSIONS: Pathologic examination of regional lymph nodes in pancreatic adenocarcinoma specimens fails to detect metastases in many patients. Lymph node micrometastasis is one reason for the poor survival rates observed among patients with Stage I cancers. PCR/RFLP may have a role in staging early pancreatic cancers.