Saliva, diagnostics, and dentistry.

Saliva, a scientific and clinical entity familiar to every oral health researcher and dental practitioner, has emerged as a translational and clinical commodity that has reached national visibility at the National Institutes of Health and the President's Office of Science and Technology. "Detecting dozens of diseases in a sample of saliva" was issued by President Obama as one of the 14 Grand Challenges for biomedical research in the 21(st) Century (National Economic Council, 2010). In addition, NIH's 2011 Government Performance Report Act (GPRA) listed 10 initiatives in the high-risk long-term category (Collins, 2011).

Multi-Site PCR-based CMV viral load assessment-assays demonstrate linearity and precision, but lack numeric standardization: a report of the association for molecular pathology.

Viralload (VL) assessment of cytomegalovirus (CMV) by real-time PCR is an important tool for diagnosing and monitoring CMV viremia in patients with compromised immune systems. We report results from a sample exchange organized by members of the Association for Molecular Pathology that compared PCR results from 23 laboratories; 22 such laboratories used a laboratory-developed real-time PCR assay and one laboratory used a competitive PCR assay. The samples sent to each laboratory were comprised of a dilution panel of CMV virion-derived reference materials that ranged from 0 to 500,000 copies/ml.

Results of the 2001 AcroMetrix HIV-1 resistance proficiency program.

Background: Human immunodeficiency virus type 1 (HIV-1) drug resistance mutation testing has become a useful tool in assessing antiretroviral treatment and managing patient care. As these complex molecular genotyping procedures move into routine use in clinical laboratories, the need arises for quality control procedures that will ensure that drug resistance associated mutations are accurately identified.

Objectives: The AcroMetrix HIV-1 Resistance Proficiency Program was designed to assess proficiency in the identification of HIV-1 drug resistance mutations using molecular genotyping methods.

Standardized hepatitis C virus RNA panels for nucleic acid testing assays.

Background: Methods for the quantification of hepatitis C virus (HCV) RNA are useful in the clinical management of infected patients. However, the introduction of assays based upon various nucleic acid testing (NAT) technologies, each utilizing a different set of standards, creates the potential for misinterpretation of patient results.

Objective: In order to address the need for worldwide standardization of these assays, a HCV RNA quantification panel (NAP HCV-RNA) calibrated against the World Health Organization (WHO) First International Standard for HCV RNA was prepared.

Detection of hepatitis C after liver transplantation. Four serologic tests compared.

To determine the best method for detecting HCV infection in immunosuppressed patients, stored frozen serum from 101 liver transplant recipients was tested for hepatitis C virus. Each sample was tested by four assays. HCV RNA was detected by both polymerase chain reaction (PCR) and branched DNA signal amplification.

Quantitation of HBV DNA in human serum using a branched DNA (bDNA) signal amplification assay.

The aim of this study was to establish the performance characteristics of a nonradioisotopic branched DNA (bDNA) signal amplification assay for quantitation of hepatitis B virus (HBV) DNA in human serum. Quantitation was determined from a standard curve and expressed as HBV DNA equivalents/mL (Eq/mL; 285,000 Eq = 1 pg of double stranded HBV DNA). The bDNA assay exhibited a nearly four log dynamic range of quantitation and an analytical detection limit of approximately 100,000 Eq/mL.

Evaluation of branched DNA signal amplification for the detection of hepatitis C virus RNA.

There is an increasing need for a practical assay to measure HCV RNA to assess the viral burden in chronic hepatitis C virus (HCV) infection as viral load relates to transmission and therapeutic response. This study evaluates branched DNA (bDNA) signal amplification, a technique that avoids many of the pitfalls of polymerase chain reaction (PCR). The bDNA assay uses a microtitre well format and a series of capture, target and amplification probes that bind RNA to the well and then successively bind oligonucleotides to the RNA and branched DNA molecules to the oligonucleotides.

Comparison of quantitative cDNA-PCR with the branched DNA hybridization assay for monitoring plasma hepatitis C virus RNA levels in haemophilia patients participating in a controlled interferon trial.

The branched DNA (bDNA) assay was compared with a semi-quantitative cDNA-polymerase chain reaction (cDNA-PCR) assay for monitoring HCV RNA levels in plasma in 17 haemophilia patients participating in a controlled alpha-interferon trial. Good correlation between the HCV RNA levels as detected by the two assays was observed, with a correlation co-efficient of 0.83 (P < 0.

Quantitative detection of hepatitis C virus RNA with a solid-phase signal amplification method: definition of optimal conditions for specimen collection and clinical application in interferon-treated patients.

To determine the optimal conditions for preparation of serum specimens for quantitative hepatitis C virus RNA determination, patient samples were processed such that differences in time from clot formation to centrifugation, centrifugation to separation of serum and collection of serum until freezing could be independently assessed. The effects of multiple cycles of freezing and thawing were also determined. There was progressive and significant loss of hepatitis C virus RNA activity when the time from the formation of the clot until centrifugation was longer than 2 hr.

Quantitation of hepatitis C virus RNA in liver transplant recipients.

Background/aims: Hepatitis C virus (HCV) infection is common in liver transplant recipients, yet the effects of immunosuppression on HCV RNA levels and the relationship of HCV RNA levels to hepatic damage have not been studied.

Methods: To explore these issues, we measured HCV RNA in serum by polymerase chain reaction amplification and branched DNA assay from 100 HCV-infected patients undergoing liver transplantation.

Results: Mean posttransplant levels were 16-fold higher than pretransplant values (7,935,000 and 496,000 Eq/mL, respectively; n = 65; P < 0.

Quantitative evaluation of hepatitis C virus RNA in patients with concurrent human immunodeficiency virus infections.

Quantitation of the hepatitis C virus (HCV) provides a powerful epidemiologic and therapeutic method for the evaluation of infected patients. In this study semiquantitative reverse transcriptase polymerase chain reaction (PCR) is compared with a new branched DNA signal amplification methodology. Samples from HCV-infected patients as well as from human immunodeficiency virus-infected patients were evaluated.

Significance of serum hepatitis C virus RNA levels in chronic hepatitis C.

Hepatitis C virus (HCV) is the main cause of parenteral non-A, non-B hepatitis and serum can be tested for the virus itself by reverse-transcription polymerase chain amplification. What of the level of this viraemia? To find out if quantitative study of HCV RNA might be useful clinically we took advantage of participation in trials of interferon-alpha in patients with chronic HCV infection and applied a new assay, branched DNA (bDNA) signal amplification. Paired serum and liver biopsy specimens from 47 patients with confirmed chronic HCV infection and evidence of HCV RNA in their serum were studied.

Altered form of placental alkaline phosphatase produced by JAR choriocarcinoma cells in culture.

The alkaline phosphatase activity expressed by JAR choriocarcinoma cells was compared to the placental isoenzyme of human alkaline phosphatase by several criteria. JAR cell alkaline phosphatase was similar to the placental isoenzyme with respect to heat and urea stability and sensitivity to most inhibitors, but it differed significantly from placental alkaline phosphatase in its sensitivity to L-phenylalanylglycylglycine. In contrast to the serum form of placental alkaline phosphatase, the JAR cell enzyme was highly hydrophobic as determined by octyl agarose chromatography and appeared to be larger than the placental isoenzyme based on gel filtration and polyacrylamide gel electrophoresis.

Expression of oncodevelopmental gene products by human tumor cells in culture.

Sixty-seven human tumor cell lines and 15 lines derived from normal tissue were examined for the production of the oncodevelopmental markers carcinoembryonic antigen, alpha and beta subunits of chorionic gonadotropin, placental and nonplacental forms of alkaline phosphatase, gamma-glutamyltransferase, cystyl aminopeptidase, and calcitonin. Both intracellular and extracellular levels of these markers were determined at three phases during the growth of each culture. Sixty-eight percent of the cell lines produced elevated levels (greater than or equal to 90th percentile) of at least one marker.

Content of gonadotropins in cultured human malignant cells and effects of sodium butyrate treatment on gonadotropin secretion by HeLa cells.

Twenty-one of 82 human cell lines examined for production of human chorionic gonadotropin and its subunits (HCG-alpha and HCG-beta) produced either one or both subunits at some phase in their growth. Of these, 14 produced an excess amount of free alpha subunit, and seven produced HCG-beta or complete HCG without evidence for free alpha subunit synthesis. Five of the HCG-producing cell lines also contained or secreted the beta subunit of human luteinizing hormone.

Early gene expression of adenovirus type 2: R-loop mapping of mRNA and time course of viral DNA, mRNA, and protein synthesis.

Adenovirus type 2 DNA was hybridized to early mRNA isolated from the cytoplasm of infected cells prior to the initiation of viral DNA synthesis. Resulting R loops were visualized in the electron microscope, and their positions were oriented with the help of DNA fragments generated by digestion with the restriction endonuclease BamHI. Early RNA was found to map (in order of relative R-loop frequency) with midpoints near positions 0.

Electron microscopy of late adenovirus type 2 mRNA hybridized to double-stranded viral DNA.

R loops were generated with late adenovirus type 2 (Ad2) mRNA in double-stranded viral DNA, and visualized by electron microscopy. Unpaired DNA sequences in Ad2:Ad2+ND4 heteroduplex DNA served as a visual marker for the orientation of R loops with respect to the conventional DNA map. The most abundant classes of late Ad2 mRNA observed by this technique hybridized, in order of R-loop frequency, with midpoints near posit1ons 0.

In Vitro system for the study of bacteriophage phi chi 174 adsorption and eclipse.

An in vitro system was developed for the study of the initial stages of bacteriophage phi chi 174 infection. Escherichia coli C cells were incubated with 20% sucrose and then subjected to cold osmotic shock in 5 mM MgSO4. The concentrated supernatant shock fluid inactivated phi chi 174 with the same kinetics and requirements as for normal infection.