Nested Polymerase Chain Reaction Approach Research Articles

Association Between Epidermodysplasia Verruciformis-Associated Human Papillomavirus DNA in Plucked Eyebrow Hair and Solar Keratoses

Association Between Epidermodysplasia Verruciformis-Associated Human Papillomavirus DNA in Plucked Eyebrow Hair and Solar Keratoses

Prevalence of the factor VR506Q mutation in two Irish control populations: use of a novel nested polymerase chain reaction approach

The prevalence of factor V (FV) Leiden among normal populations has primarily been determined using blood donors. This control group is carefully selected and therefore may not accurately reflect the true prevalence within the population. We assessed the prevalence of FV Leiden within the Irish population using Guthrie card samples randomly selected from all newborns. We compared this result with the prevalence of FV Leiden within blood donors. A novel nested polymerase chain reaction (PCR) method for FV Leiden was developed for analysis of the Guthrie card samples. There was no significant difference between the allele frequency within the Guthrie card samples and blood donors (2·07% vs. 2·35%, P = 0.66)

Development and validation of a multiplex PCR-based assay for the upper respiratory tract bacterial pathogens haemophilus influenzae, streptococcus pneumoniae, and moraxella catarrhalis

Background: Conventional simplex polymerase chain reaction (PCR)-based assays are limited in that they only provide for the detection of a single infectious agent. Many clinical diseases, however, present in a nonspecific, or syndromic, fashion, thereby necessitating the simultaneous assessment of multiple pathogens. Panel-based molecular diagnostic testing can be accomplished by the development of multiplex PCR-based assays, which can detect, individually or severally, different pathogens that are associated with syndromic illness. As part of a larger program of panel development, an assay that can simultaneously detect Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis was developed. These organisms were chosen as they are the most common bacterial pathogens associated with both the acute and chronic forms of otitis media; they are also responsible for a high percentage of sinus infections in both children and adults. In addition, H. influenzae and S. pneumoniae are commonly associated with septic meningitits. Methods and Results: Multiple individual PCR-based assays were developed for each of the three target organisms which were then evaluated for sensitivity and specificity. Utilizing the simplex assays that met our designated performance criteria, a matrix style approach was used to develop a duplex H. influenzae-S. pneumoniae assay. The duplex assay was then used as a single component in the development of a triplex assay, wherein the various M. catarrhalis primer-probe sets were tested for compatibility with the existing assay. A single-step PCR protocol, with species-specific primers for each of the three target organisms and a liquid hybridization-gel retardation amplimer detection system, was developed, which amplifies and then discriminates among each of the amplification products according to size. This assay is able to detect all three organisms in a specific manner, either individually or severally. Dilutional experiments indicate a detection limit of 10 femtograms (fg)(6-7 genomic equivalents) or less of genomic DNA for each of the three microorganisms regardless of the presence of irrelevant DNA. Conclusions: The reliance on individual, robust, species-specific primers and the avoidance of a nested PCR approach make this bacterial multiplex assay suitable for use in the clinical laboratory. This assay has proved useful in both research and patient care applications. (Mol Diagn 1996 Jun;1(1):29-39)

Detection of Enterotoxigenic Clostridium perfringens in Raw Beef by Polymerase Chain Reaction

A polymerase chain reaction (PCR) procedure was developed for direct detection of Clostridium perfringens strains with potential for food poisoning in raw beef samples. An oligonucleotide primer pair was used to amplify a 364 base pair sequence internal to the C. perfringens enterotoxin gene. One milliliter portions of the meat homogenates were inoculated into cooked meat medium (CMM) or reduced Fluid Thioglycollate (FTG) medium and incubated at 37°C. Portions sampled at 2, 4, 6, 8 and 24 h of enrichment were assayed for detection of the enterotoxin sequence by PCR. Amplification of the 364 bp sequence could be detected in 6 h by agarose gel electrophoresis and as early as 2 h by hybridization to a 150 bp digoxigenin (DIG)-labeled probe. To increase the sensitivity of the detection assay a commercial chromosomal deoxyribonucleic acid (DNA) extraction assay was compared with a nested PCR approach. Both methods allowed detection of less than 1 log10 colony forming units (CFU)/g of C. perfringens strains harboring the enterotoxin gene, with no interference with the background microflora present in the raw ground beef.

Molecular analysis of IgE H-chain transcripts expressed in vivo by peripheral blood lymphocytes from normal and atopic individuals.

The low levels of IgE produced by PBMC from normal individuals has so far prevented an analysis of their IgE H chain repertoire. Using a nested polymerase chain reaction approach, we were able to detect epsilon transcripts in all normal and allergic individuals we investigated. We further cloned epsilon CDR3/FW4 regions from two normal and two atopic individuals with low serum IgE levels. Sequence analysis of 104 clones identified 26 different epsilon CDR3/FW4 regions and an additional number of clonally related transcripts in the two atopic individuals. Preferential usage of DH genes from the DXP family (33%) and of the JH4b gene (35%) were observed, similar to reported findings for the IgM-producing peripheral blood B cell subset. Using CDR3 specific oligonucleotides, we detected the CDR3/FW4 regions of a particular set of clonally related epsilon transcripts in mu and gamma 4 transcripts of the same individual. This finding demonstrates the in vivo production of IgE together with the two other Ig isotypes (IgM and IgG4) by the progeny of a common B cell precursor, and suggests a possible mechanism for regulating the allergic response. The clonally related epsilon transcripts were found to be only of the secreted form. We give also evidence that the IgE-producing B cells undergo somatic mutation because a number of identical mutations were observed in the FW4 regions of epsilon and mu clonally related transcripts. Some of these mutations were shared with other transcripts from the same and other individuals, supporting the existence of sequence-specific hot spots for the somatic hyper-mutation machinery in the JH gene segments.