A colorimetric one-tube nested PCR was developed for the detection of Trichomonas vaginalis in clinical vaginal discharge specimens. A family of 650-bp specific DNA repeats from the T. vaginalis genome was targeted. There was no cross-reaction with human DNA or other infectious agents, including Pentatrichomonas hominis and Giardia lamblia. The colorimetric assay was applied as an adjunct to ne...

Structural alterations of the apolipoprotein E (apo E) are known to influence the lipid metabolism. The three most common isoproteins Arg 158) are encoded by three co-dominant alleles (e2, el, e4). Phenotyping is cumbersome and, therefore, several methods for apo E genotyping have been developed. Of these, PCR-RFLP (restriction fragment length polymorphism) is currently most rapid and cost-effe...

With increasing knowledge about the causal role of genetic defects in clinical diseases the necessity is apparent to have procedures for rapid diagnosis of point mutations. We developed a PCR-based technique, whereby both normal and mutant alleles can be amplified in the same reaction tube, using different length allele-specific primers. Furthermore the allele-specific primers introduce additio...

INTRODUCTION Herein, we report a one-tube, semi-nested-polymerase chain reaction (OTsn-PCR) assay for the detection of Paracoccidioides brasiliensis. METHODS We developed the OTsn-PCR assay for the detection of P. brasiliensis in clinical specimens and compared it with other PCR methods. RESULTS The OTsn-PCR assay was positive for all clinical samples, and the detection limit was better or ...

Functional genomics requires vector construction for protein expression and functional characterization of target genes; therefore, a simple, flexible and low-cost molecular manipulation strategy will be highly advantageous for genomics approaches. Here, we describe a Ω-PCR strategy that enables multiple types of sequence modification, including precise insertion, deletion and substitution, in ...

A rapid method for efficiently generating site-directed mutations on a clean sequence background is described. This modification of the megaprimer PCR mutagenesis approach can be performed in one tube in less than 4.5 hours, and does not require purification of intermediate products. High fidelity of DNA sequence replication is obtained by employing Pfu DNA polymerase and limiting the total num...

A multiplex PCR (m-PCR) with primers targeting the 16S rRNA, Rv3873 and a 12.7-kb fragment in the genomes of a Mycobacterium tuberculosis complex was designed for the differential diagnosis of M. tuberculosis, M. bovis, M. bovis BCG and non-tuberculosis Mycobacterium (NTM). The specificity of this assay was 100%, and the detection limit was 15 pg of genomic DNA. Of the 206 blinded clinical samp...

There is a growing appreciation of the potential value for routine screening for the presence of HPV not only for cervical specimens but also from oral cavity. The purpose of this study was to develop and clinically evaluate a single-tube seminested PCR assay for the detection of HPV. Several parameters such as PCR primers, primer annealing temperature, the number of PCR cycles and concentratio...

Classical swine fever (CSF) is a notifiable, highly contagious disease of swine controlled mainly with costly administrative methods. Swine may be infected not only with classical swine fever virus (CSFV), but also with other, non porcine, genetically and antigenically related pestiviruses. Differentiation of infections with CSFV and other pestiviruses is a crucial element of diagnostics. In th...