Optimized PCR amplification of influenza A virus RNA using Tth DNA polymerase,incorporating uracil N glycosylase (UNG) in a single tube reaction

An optimized reaction condition for amplification of influenza A virus RNA, by thermus thermophilus (Tth) DNA polymerase-based PCR, incorporating uracil N glycosylase (UNG) and dUTP in the reaction has been determined. DUTP could not be substituted for all dTTP sites when UNG was present in the reaction. The relative concentration of dUTP and dTTP has been optimized for allowing amplification of the target RNA. It has been verified that the amplified product DNA had sufficient dUTP and was digestable by UNG. Using the optimized reaction condition, influenza A virus-specific DNA fragment could be amplified and detected in 15 of 15 culture positive (for influenza A virus) nasopharyngeal specimens. J. Clin. Lab. Anal. 11:323–327, 1997. © 1997 Wiley-Liss, Inc.     

Use of xenogenized (modified) tumor cells for treatment in experimental tumor and in human neoplasia.

The need to modify tumor cells in order to render them more "immunogenic" was based on the assumption that normal, nonmodified tumor cells are non- or weakly immunogenic and as such are unable to raise an efficient protective immune response. Various methods for "xenogenization" (modification of tumor cells) were suggested: induction of new foreign antigens, treatment with either chemicals or enzymes and use of mutagens. Xenogenized tumor cells by their coupling to proteins, and use of chemicals like DTIC (5-[3,3-dimethyl- 1-triazeno]-imidazole-4-carboxamide), TZC (8-carbamoyl-3-methyl-imidazo[5, 1-d]- 1,2,3,5-tetrazin-4 [3H]-one 8-carbamoyl-3-[2-chloroethyl] imidazole [5,1 -d]- 1,2,3,5-tetrazin-4[3H]-one) and antiemetic drugs, were tested in experimental models of murine leukemia. Non-tumorigenic clones, xenogenization with DNA hypomethylating agents, aryl-triazine derivatives and DTIC were evaluated for their induction of protective immune response in murine lymphoma. Murine plasmacytoma cells were used for immunization after treatment with glutaraldehyde. Viral modifications of tumor cells were evaluated for their ability to induce a protective tumor response in model systems of rat fibrosarcoma, liver metastatic rat tumor cells, lymphoid tumor cells and hamster tumor cells. In the case of human cancer, attempts were reported to use DNP-conjugated melanoma cells, mutagenic triazine compounds, an autologous colon tumor cell bacillus Calmette-Guerin (BCG) vaccine and genetically engineered vaccines for immunization. The general conclusion drawn from experimental tumor models and for human cancer is, that although modified tumor cells were found to be partially effective in experimental models, it is still necessary to provide more data in order to determine the effective use of xenogenized human tumor cells for immunotherapy.     

丙型肝炎病毒核酸检测的国家标准物质的研制

目的研制国内用于丙型肝炎病毒核酸(HCVRNA)扩增检测的血清标准物质。方法用HCVRNA阴性血浆将阳性血浆稀释至含量约300000拷贝数/ml,然后进行真空冷冻干燥。检测方法采用实时荧光定量聚合酶链反应方法和罗氏公司的PCR内标定量方法。制备标准品含量通过与国际标准(世界卫生组织属下的国家生物标准研究所标准物质编号:96/790)比对获得。结果该标准物质定值为:(1·29±0·24)×105IU/ml。其稳定性实验表明室温20~25℃(相对湿度20%~50%)14d、37℃(相对湿度60%~80%)7d均稳定。长期监测结果表明:2~8℃6个月及-20℃保存两年的含量均无明显变化。均一性检测结果:瓶间不精密度为3·53%。结论该制备物可以作为用于核酸扩增检测的丙型肝炎病毒核酸国家标准物质。     

Analytic performance and contamination control methods of a ligase chain reaction DNA amplification assay for detection of Chlamydia trachomatis in urogenital specimens

A ligase chain reaction (LCR) DNA amplification assay that targeted the cryptic plasmid of Chlamydia trachomatis was developed to detect C. trachomatis urogenital tract infection. The objectives of this study were to determine the cutoff and analytic performance of the LCR assay and to characterize the effectiveness of its postdetection contamination control method. The assay's cutoff was determined after receiver-operator characteristic (ROC) analysis of 4660 clinical data points. The assay detected one infectious unit per reaction of each of the 15 C. trachomatis serovars and did not cross-react with 13 Chlamydia pneumoniae strains, 13 Chlamydia psittaci strains, and 87 other bacteria, fungi, parasites, or viruses. In addition, the assay did not detect 77 processed urine specimens collected from patients with urinary tract infections caused by yeast or bacteria other than C. trachomatis. The assay was sufficiently precise to detect consistently two infectious units of C. trachomatis per reaction. False-positive assay results attributable to contamination with amplified product were minimized by the use of standard procedures as well as by a postdetection chemical inactivation method that could reduce the amount of amplified LCR product by a factor of ⩾10⁷.     

Enzymatic amplification of platelet-specific messenger RNA using the polymerase chain reaction.

Human platelets are derived from megakaryocytes as anucleate cells, and thus contain only vestigial amounts of RNA capable of being transcribed into protein. This has greatly hampered efforts to study directly platelet-specific gene products and their associated polymorphisms. In this report, we describe direct amplification, using the polymerase chain reaction, of platelet-derived mRNA in amounts sufficient to permit detailed analysis, such as restriction mapping and nucleotide sequencing. The ability to generate large amounts of cDNA from platelet-specific mRNA sequences should make possible direct molecular characterization of normal platelet proteins, and facilitate the investigation of a wide variety of inherited platelet disorders.     

Simultaneous amplification of DNA and RNA virus using multiplex PCR system.

A large number of disease-causing bacteria and viruses are being sequenced and PCR is increasingly used for the diagnosis of the diseases. We have designed a multiplex PCR system for hepatitis B virus (HBV), a DNA virus, and hepatitis E virus (HEV), an RNA virus. A modified technique has been standardized for simultaneous extraction of DNA and RNA, followed by a one-step RT-PCR/PCR.     

Use of a PCR-based approach for sequencing whole mitochondrial genomes of insects: two examples (cockroach and dragonfly) based on the method developed for decapod crustaceans

Recent development of a PCR-based approach for sequencing vertebrate mitochondrial genomes has attracted much attention as being more rapid and economical than traditional methods using cloned mtDNA and primer walking. Such a method has not been available for insect mitochondrial genomes, despite widespread use of them for the molecular phylogenetic, biogeographical and population genetic markers. A recently developed PCR-based approach for sequencing whole mitochondrial genomes of decapod crustaceans, which included the design of many versatile PCR primers for the latter, was applied with the same primers sets to mitochondrial genomes of two insects, smoky-brown cockroach Periplaneta fuliginosa (Serville, 1839) and skimmer dragonfly Orthetrum triangulare melania (Selys, 1883). Almost the entire region of the two mitochondrial genomes was successfully sequenced. Features of the two mitochondrial genomes are described and the usefulness of this PCR-based approach for sequencing insect mitochondrial genomes demonstrated.     

Use of PCR amplification for diagnosis of HTLV-I infection after blood transfusion.

We studied the seroconversion to human T-cell leukemia virus type-1 (HTLV-I) in two immunocompromised patients after transfusions of cellular blood components. One patient produced IgM antibodies against the viral p19 protein 149 days post-transfusion (a serum on day 43 was negative). Both patients showed indeterminate Western-blots (IgG anti-p19 and anti-gp46 but no anti-p24). Using the polymerase chain reaction (PCR) with two primer pairs (SK43/44 and SK54/56), we demonstrated HTLV-I infection prior to seroconversion. This infection was confirmed by Southern blot.     

A modified thrombin clotting time test as a quality control marker for heparin contamination in obstetric intraoperative cell salvage

Objective: To assess if a modified thrombin clotting time test could be used as a simple quality control (QC) method to screen for unfractionated heparin in the product obtained from obstetric intraoperative cell salvage cases before re‐infusion. Background: A national QC scheme has recently been piloted to monitor the quality of autologous blood being returned to the patient. Laboratory tests include full blood count and microalbumin. Unfractionated heparin testing should be performed to ensure that there is no gross contamination of heparin in the final product; however, presently, there is no quick cheap test available suitable for heparin detection. Materials and Methods: Samples were collected into plain non‐anticoagulated tubes and centrifuged at 2500 ×g for 5 min. Supernatant was mixed with commercially available coagulated normal plasma and a thrombin clotting time test performed. Results: Calibration runs demonstrated that our system was sensitive up to 0·14 IU mL^?1 heparin, linear between 0·08 and 0·14 IU mL^?1. Conclusion: We have shown that the thrombin clotting time test can be modified and used as a cheap and reliable marker for heparin contamination. We have successfully incorporated this modified test into our hospital's obstetric QC scheme.     

Covergowns and the control of operating room contamination

This study assessed the effectiveness of cotton/polyester covergowns in protecting scrubsuits against bacterial contamination when operating room (OR) personnel are outside the clean environment of the operating suite. Rodac impression plates were used to measure bacterial contamination. The subjects were nurses working a normal daily OR routine. Bacterial colony counts on the right shoulder decreased when covergowns were worn over scrubsuits during the lunch period outside the OR and when fresh scrubsuits were put on following the lunch period. Colony counts rose over the lunch period when scrubsuits were worn unprotected outside the OR and when scrubsuits were removed before and put on again following lunch. Left thigh samples showed no significant effects of experimental treatments and yielded a mean colony count 2.8 times higher than right shoulder samples. Fifty-three percent of subjects were positive for Staphylococcus aureus and 16% yielded positive plates on 3 or more study days. The incidence of S. aureus contamination was affected by experimental treatments in a way similar to overall bacterial contamination. The results indicated that wearing covergowns protects against above-waist bacterial contamination of scrubsuits.     

Simultaneous allele-specific amplification: a strategy using modified primer-template mismatches for SNP detection--application to prothrombin 20210A (factor II) and factor V Leiden (1691A) gene mutations.

BACKGROUND: Inherited thrombophilia is caused by mutations in genes central to the clotting cascade. Analysis of the factor V Leiden (FVL) and prothrombin G20210A mutations are the most prevalent in thrombophilia. METHODS AND RESULTS: We have optimized an allele-specific PCR assay for the simultaneous detection of both wild-type and mutant alleles. This method is adapted for clinical use with the FVL and prothrombin G20210A assays and is significant in its intentional use of nucleotide mismatches at the 3' end of allele-specific primers. Two internal allele-specific primers are designed to amplify in opposite directions on opposite strands that reduce differential amplification. Our results show concordance with methods involving PCR with restriction endonuclease digestion, yet are simpler to perform. CONCLUSION: The simultaneous allele-specific amplification method allows simultaneous detection of wild-type and mutant alleles by PCR using four distinct primers. Nucleotide mismatches in the primers reduce competitive amplification.     

Use of DNA amplification methods for clinical diagnosis in autoimmune diseases

Autoimmune disease is generally felt to result from the interaction of genetic and environmental factors. In recent years, significant advances have been made in using recombinant DNA methods to analyze specific genetic factors and infectious agents. However, new techniques are needed that are more rapid, inexpensive, and suitable for small tissue biopsies obtained early in the course of disease. New methods of DNA amplification based on polymerase chain reaction (PCR) and Q beta-replicase (Q beta R) have recently been reported. These methods are briefly reviewed, and their potential applications to patients with autoimmune disease are presented. Several types of applications can be considered, including detection of: a) specific HLA-D alleles in order to predict prognosis and better utilize existing medications; b) bacterial, fungal, and spirochete infections in joint aspirates or synovial biopsies; c) human immunodeficiency virus (HIV) and other viruses (e.g., EBV, CMV) that may be associated with immune dysregulation in certain patients; and d) neoplastic transformation in blood or tissues by determining monoclonal gene rearrangements, karyotypic alterations or oncogene activation. It is likely that routine clinical laboratories will soon begin implementing DNA amplification methods in order to screen blood products for infectious agents (especially HIV and hepatitis B virus). Because these techniques will be readily available, rheumatologists/clinical immunologists should begin developing strategies that will allow them to use these methods in a cost-effective manner for diagnosis and monitoring treatment.     

Leishmania (Viannia) subgenus kDNA amplification for the diagnosis of mucosal leishmaniasis

The utility of 2 polymerase chain reaction (PCR)-based assays amplifying genus or Viannia subgenus Leishmania minicircle kDNA for the diagnostics of ML was assessed. The Viannia subgenus product was yielded after PCR from isolates of L. (Viannia) braziliensis, L. (Viannia) colombiensis, and L. (Viannia) guyanensis, whereas no product was obtained with the non-Viannia-pertaining species: L. (Leishmania) amazonensis, L. (Leishmania) donovani, and L. (Leishmania) chagasi. With both assays, 11 of 13 (86.4%) patients with confirmed ML could be identified, whereas only 2 (16.7%) of these patients were positive by microscopy. All amplified genus-specific products gave a positive signal by hybridization with a Leishmania (Viannia) subgenus-specific radioactive probe. The Viannia subgenus-specific kDNA PCR represents a sensitive and specific tool for the diagnosis of ML, remarkably improving the sensitivity of parasitological methods and offering an alternative for the radioactive-dependent assays for subgenus characterization.     

Use of equilibrated blood for internal blood-gas quality control.

We have used equilibrated human blood for blood-gas quality control since 1970. In blood equilibrated 24 h after shedding, gas tensions are stable for 4 to 6 h at 0 to 4 degrees C; each control specimen is analyzed several times during that period to resolve malfunctions, etc. Three-fourths of all errors in gas-tension measurement detected with equilibrated blood were detected with the highest-tension controls. Equilibrated blood controls signal about one error every 14 d on each instrument. For more complete quality control, we supplement analysis of equilibrated blood with other sorts of controls, comparing results obtained by assaying each patient's specimen on two instruments being our most effective adjunct. Such comparisons have identified erroneous assays in 3.9% of the specimens tested. The magnitude of interinstrument discrepancies (random errors) have ranged from 9 to 100% of the appropriate determinations. We use control data derived from equilibrated blood analysis for special management purposes (evaluating instruments, quantitating micro- vs. macro-sampling discrepancies, and decreasing instrument-repair costs).