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Amplitaq and Amplitaq Gold DNA Polymerase AmpliTaq and AmpliTaq Gold DNA Polymerase The Most Referenced Brand of DNA Polymerase in the World Date: 2005-05 Notes: Authors are listed alphabetically J. Clin. Microbiol. (237) Beck, I. A., M. Mahalanabis, et al. (2002). "Rapid and Sensitive Oligonucleotide Ligation Assay for Detection of Mutations in Human Immunodeficiency Virus Type 1 Associated with High-Level Resistance to Protease Inhibitors." J. Clin. Microbiol. 40(4): 1413-1419. http://jcm.asm.org/cgi/content/abstract/40/4/1413 A sensitive, specific, and high-throughput oligonucleotide ligation assay (OLA) for the detection of genotypic human immunodeficiency virus type 1 (HIV-1) resistance to Food and Drug Administration-approved protease inhibitors was developed and evaluated. This ligation-based assay uses differentially modified oligonucleotides specific for wild-type or mutant sequences, allowing sensitive and simple detection of both genotypes in a single well of a microtiter plate. Oligonucleotides were designed to detect primary mutations associated with high-level resistance to amprenavir, nelfinavir, indinavir, ritonavir, saquinavir, and lopinavir, including amino acid substitutions D30N, I50V, V82A/S/T, I84V, N88D, and L90M. Plasma HIV-1 RNA from 54 infected patients was amplified by reverse transcription-PCR and sequenced by using dideoxynucleotide chain terminators for evaluation of mutations associated with drug resistance. These same amplicons were genotyped by the OLA at positions 30, 50, 82, 88, 84, and 90 for a total of 312 codons. The sensitivity of detection of drug-resistant genotypes was 96.7% (87 of 90 mutant codons) in the OLA compared to 92.2% (83 of 90) in consensus sequencing, presumably due to the increased sensitivity of the OLA. The OLA detected genetic subpopulations more often than sequencing, detecting 30 mixtures of mutant and wild-type sequences and two mixtures of drug- resistant sequences compared to 15 detected by DNA sequencing. Reproducible and semiquantitative detection of the mutant and the wild-type genomes by the OLA was observed by analysis of wild-type and mutant plasmid mixtures containing as little as 5% of either genotype in a background of the opposite genome. This rapid, simple, economical, and highly sensitive assay provides a practical alternative to dideoxy sequencing for genotypic evaluation of HIV-1 resistance to antiretrovirals. Collantes-Fernandez, E., A. Zaballos, et al. (2002). "Quantitative Detection of Neospora caninum in Bovine Aborted Fetuses and Experimentally Infected Mice by Real-Time PCR." J. Clin. Microbiol. 40(4): 1194-1198. http://jcm.asm.org/cgi/content/abstract/40/4/1194 We report the development of a real-time PCR assay for the quantitative detection of Neospora caninum in infected host tissues. The assay uses the double-stranded DNA-binding dye SYBR Green I to continuously monitor product formation. Oligonucleotide primers were designed to amplify a 76-bp DNA fragment corresponding to the Nc5 sequence of N. caninum. A similar method was developed to quantify the 28S rRNA host gene in order to compare the parasite load of different samples and to correct for the presence of potential PCR-inhibiting compounds in the DNA samples. A linear quantitative detection range of 6 logs with a calculated detection limit of 10-1 tachyzoite per assay was observed with excellent linearity (R2 = 0.998). Assay specificity was confirmed by using DNA from the closely related parasite Toxoplasma gondii. The applicability of the technique was successfully tested in a variety of host brain tissues: (i) aborted bovine fetuses classified into negative or positive Neospora-infected animals according to the observation of compatible lesions by histopathological study and (ii) experimentally infected BALB/c mice, divided into three groups, inoculated animals with or without compatible lesions and negative controls. All samples were also tested by ITS1 Neospora nested PCR and a high degree of agreement was shown between both PCR techniques ({kappa} = 0.86). This technique represents a useful quantitative diagnostic tool to be used in the study of the pathogenicity, immunoprophylaxis, and treatment of Neospora infection. Gray, J., L. V. von Stedingk, et al. (2002). "Transmission Studies of Babesia microti in Ixodes ricinus Ticks and Gerbils." J. Clin. Microbiol. 40(4): 1259-1263. http://jcm.asm.org/cgi/content/abstract/40/4/1259 In order to investigate the possible role of Ixodes ricinus as a vector of zoonotic Babesia microti infection in Europe, a European rodent isolate (HK) and a zoonotic American isolate (GI) were studied in transmission experiments. PCR detected B. microti in the blood and spleens of infected gerbils (Meriones unguiculatus) and also in laboratory-induced infections of I. ricinus ticks. B. microti DNA was detected by PCR in all pooled samples of nymphs and the majority of adults that had fed as larvae and nymphs, respectively, on gerbils with acute infection of the European isolate, confirming that I. ricinus could serve as a vector in Europe. The American isolate, GI, proved to be equally infective for larval and nymphal I. ricinus as the HK strain, despite a very different appearance in gerbil erythrocytes. Nymphs infected with the HK and GI strains readily infected gerbils. In contrast to the finding in acute infections, ticks that fed on gerbils with chronic infections of HK and GI did not become infected. It was also found that the HK strain was not transmitted transovarially. The finding that a B. microti strain (GI) from a distant geographical region (United States) can infect and be transmitted by I. ricinus suggests that other European B. microti strains, in addition to the HK strain used here, are probably infective for I. ricinus, supporting the view that infection of humans with European B. microti may be a regular occurrence. Leary, T. P., J. C. Erker, et al. (2002). "Detection of Mammalian Reovirus RNA by Using Reverse Transcription-PCR: Sequence Diversity within the {lambda}3-Encoding L1 Gene." J. Clin. Microbiol. 40(4): 1368-1375. http://jcm.asm.org/cgi/content/abstract/40/4/1368 Reoviruses infect virtually all mammalian species, and infection of humans is associated with mild gastrointestinal or upper respiratory illnesses. To improve reovirus detection strategies, we developed a reverse transcription-PCR technique to amplify a fragment of the reovirus L1 gene segment. This assay was capable of detecting 44 of 44 reovirus field isolate strains and was sufficiently sensitive to detect nearly a single viral particle (1.16 {+/-} 0.13) per PCR of prototype strain type 3 Dearing. Pairwise comparisons of the 44 partial L1 gene sequences revealed that nucleotide variability ranged from 0 to 24.7%, with most of the nucleotide polymorphism occurring at synonymous positions. Phylogenetic trees generated from amplified L1 gene sequences suggest that multiple alleles of the L1 gene cocirculate in nature and that genetic diversity of the L1 gene is largely independent of the host species, geographic locale, or date of isolation. Phylogenetic trees constructed from the L1 gene sequences are distinct from those constructed from the four reovirus S-class gene segments, which supports the hypothesis that reovirus gene segments reassort in nature. This study establishes a new sensitive and specific technique for the identification of mammalian reoviruses and enhances our understanding of reovirus evolution. Maes, N., J. Magdalena, et al. (2002). "Evaluation of a Triplex PCR Assay To Discriminate Staphylococcus aureus from Coagulase-Negative Staphylococci and Determine Methicillin Resistance from Blood Cultures." J. Clin. Microbiol. 40(4): 1514-1517. http://jcm.asm.org/cgi/content/abstract/40/4/1514 A triplex PCR targeting the 16S rRNA, mecA, and nuc genes was developed for identification of staphylococci and detection of methicillin resistance. After validation of the assay with a collection of strains of staphylococci and enterococci (n = 169), the assay was evaluated with cultures of blood with gram-positive cocci from 40 patients. Accurate results were obtained for 59 (98%) of 61 cultures within 6 h of growth detection. Yoshida, T., T. Deguchi, et al. (2002). "Quantitative Detection of Mycoplasma genitalium from First-Pass Urine of Men with Urethritis and Asymptomatic Men by Real-Time PCR." J. Clin. Microbiol. 40(4): 1451-1455. http://jcm.asm.org/cgi/content/abstract/40/4/1451 We developed a TaqMan-based real-time PCR assay for quantifying Mycoplasma genitalium. This assay is able to specifically quantify concentrations of the M. genitalium 16S rRNA gene ranging from 107 to 10 copies/reaction. Using the TaqMan assay, we quantified the M. genitalium 16S rRNA gene in first-pass urine of men with urethritis and asymptomatic men who were positive for M. genitalium by PCR- and phylogeny-based assay. Of 130 men with gonococcal urethritis (GU), five were positive for M. genitalium. The mycoplasma load for each specimen was <5 x 10 copies/ml. Of 84 men with chlamydial non-GU (CNGU), seven were positive for M. genitalium. One man had an M. genitalium load of <5 x 10 copies/ml, and six men had loads ranging from 1.1 x 107 to 2.7 x 102 copies/ml. Of 86 men with nonchlamydial NGU (NCNGU), 17 were positive for M. genitalium. The mycoplasma loads for these men ranged from 3.3 x 106 to 2.3 x 102 copies/ml. Of 76 asymptomatic men, only two were positive for M. genitalium. For these men, the loads were 2 x 102 and <5 x 10 copies/ml. The patients with NGU had significantly higher concentrations of M. genitalium in their first-pass urine than did men with GU (P < 0.01) or asymptomatic men (P < 0.05). In addition, M. genitalium loads were significantly higher in men with NCNGU than those in asymptomatic men (P < 0.05). The quantitative assessment of M. genitalium loads by the TaqMan assay will provide useful information for understanding the pathogenicity of this mycoplasma in the urogenital tract.
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