Fluorogenic Polymerase Chain Reaction Assay Research Articles

Selective Toll-Like Receptor Expression in Human Fetal Lung

Toll-like receptors (TLRs) are critical components of the innate immune system, acting as pattern recognition molecules and triggering an inflammatory response. TLR associated gene products are of interest in modulating inflammatory-related pulmonary diseases of the neonate. The ontogeny of TLR-related genes in human fetal lung has not been previously described and could elucidate additional functions and identify strategies for attenuating the effects of fetal inflammation. We examined the expression of 84 TLR-related genes on 23 human fetal lung samples from three groups with estimated ages of 60 (57-59 d), 90 (89-91 d), and 130 (117-154 d) d. By using a false detection rate algorithm, we identified 32 genes displaying developmental regulation with TLR2 having the greatest up-regulation of TLR genes (9.2-fold increase) and TLR4 unchanged. We confirmed the TLR2 up-regulation by examining an additional 133 fetal lung tissue samples with a fluorogenic polymerase chain reaction assay (TaqMan) and found an exponential best-fit curve during the study time. The best-fit curve predicts a 6.1-fold increase from 60 to 130 d. We conclude that TLR2 is developmentally expressed from the early pseudoglandular stage of lung development to the canalicular stage.

SULFADOXINE–PYRIMETHAMINE EFFICACY AND SELECTION OF PLASMODIUM FALCIPARUM DHFR MUTATIONS IN BURKINA FASO BEFORE ITS INTRODUCTION AS INTERMITTENT PREVENTIVE TREATMENT FOR PREGNANT WOMEN

Sulfadoxine-pyrimethamine efficacy was determined with a 28-day follow-up in 97 children between 6 months and 15 years of age. The polymerase chain reaction (PCR)-corrected treatment failure was 8.2% and the uncorrected was 21.6%. The presence of the dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) mutations linked to sulfadoxine-pyrimethamine resistance before and after treatment was determined by PCR-restriction fragment length polymorphism (RFLP) and by a fluorogenic PCR assay. Before treatment, the prevalence of the triple DHFR mutations was higher among the patients having had a recurrent parasitemia (either recrudescence or new infection; 28.6% versus 9.3%), although the difference was not significant (P = 0.1). The double mutation Ala-436/Gly-437 was observed in 67% of samples, whereas no Glu-540 mutation was found. After treatment, the triple DHFR mutation was found in 76.2% of patients with recurrent parasitemia, recrudescence, and new infection alike. Such high prevalence of mutant parasites indicates that sulfadoxine-pyrimethamine should not be used as monotherapy.

Determination of Ciprofloxacin and Nalidixic Acid Resistance in Campylobacter jejuni with a Fluorogenic Polymerase Chain Reaction Assay

A fluorogenic polymerase chain reaction assay for the gyrA gene was used to determine the frequency of a Thr-86 mutation in Campylobacter jejuni isolates from food animals and humans in northern Thailand and to investigate the correlation between this mutation and bacterial resistance to fluoroquinolones. Eighty-four isolates of C. jejuni were used: 65 from healthy chickens on farms, 16 from chickens at the slaughterhouse, 1 from chicken meat at the market, and 1 from a healthy farm worker. The microbroth dilution technique was used for in vitro susceptibility testing. MIC breakpoints established by the National Antimicrobial Resistance Monitoring System were used to categorize the resistance of C. jejuni to ciprofloxacin and nalidixic acid. Sixty of the 84 C. jejuni isolates tested carried the Thr-86 mutation in the gyrA gene. All isolates with ciprofloxacin MICs of ≥ 2 mg/liter carried the mutation, and no isolates with nalidixic acid MICs of ≤16 mg/liter carried the Thr-86–to–Ile mutation. There was a very strong association between ciprofloxacin resistance and the presence of the mutation (kappa = 0.971, P < 0.01). The association between the presence of the Thr-86–to–Ile mutation and nalidixic acid resistance was weaker (kappa = 0.859; P ≤ 0.01).

Identification of Campylobacter jejuni Isolates from Cloacal and Carcass Swabs of Chickens in Thailand by a 5′Nuclease Fluorogenic Polymerase Chain Reaction Assay

A rapid 5′ nuclease fluorogenic polymerase chain reaction (PCR) assay for identifying Campylobacter jejuni was applied to Campylobacter isolates from chicken cloacal and carcass swabs collected from three chicken farms and a slaughterhouse in Thailand. The primers and the probe were based on the sequence of the gyrA gene in C. jejuni. C. jejuni isolates were identified by fluorogenic PCR assay of bacterial cells directly from Campylobacter-selective agar medium. This assay allowed the identification of C. jejuni within 1 day after colonies appeared on selective media. The fluorogenic PCR assay yielded results comparable to those of the conventional test kit (kappa = 0.76) but required less time. When the two methods disagreed with regard to species identification, results were confirmed by PCR restriction fragment length polymorphism of 23S rRNA genes. In these instances, the fluorogenic PCR assay correctly identified more isolates of C. jejuni than did the conventional test kit (six of seven isolates were unidentifiable by the conventional test kit). The fluorogenic PCR assay is a rapid and specific method that outperforms the conventional test kit in the identification of C. jejuni from environmental samples.

Detection of multiresistantSalmonella typhimuriumDT104 using multiplex and fluorogenic PCR

Salmonellainfections continue to cause gastrointestinal and systemic disease throughout the world.Salmonella typhimuriumDT104 further poses a major health concern due to its acquisition of resistance to multiple antibiotics. The rapid detection of multiresistantS. typhimuriumDT104 would facilitate strategies aimed at controlling this pathogen. We developed a specific and sensitive polymerase chain reaction (PCR) assay that amplifies a segment of DNA that is conserved in multiresistantS. typhimuriumDT104. To provide further specificity for this PCR-based diagnostic test, we amplified two other gene fragments that are present inS. typhimuriumDT104. A multiplex PCR containing primers for targeted sequences resulted in the amplification of predicted size fragments fromS. typhimuriumDT104 exhibiting the ACSSuT (ampicillin, chloramphenicol, streptomycin, sulphamethoxazole and tetracycline) or ASSuT resistance phenotypes. A minor modification of the multiplex PCR enabled the detection of other related multiresistantSalmonellasuch asS. typhimuriumU302. To augment the detection process, we also designed a fluorogenic PCR assay that can detect the DNA of multiresistantS. typhimuriumDT104 in the presence of excess contaminating bacterial DNA. These results provide a method by which multiresistantS. typhimuriumDT104, or potentially the next emerging multiresistantSalmonella, can be accurately detected in only 3–4 h.

The evaluation of a fluorogenic polymerase chain reaction assay for the detection of Salmonella species in food commodities

The TaqMan TM LS-50B PCR Detection System facilitates the automated and direct detection of polymerase chain reaction (PCR) products. The system employs the 5′ nuclease activity of Taq DNA polymerase to hydrolyse a Salmonella specific internal fluorogenic probe for monitoring the amplification of a 287-bp region of the Salmonella invA gene. Using the fluorogenic 5′ nuclease assay, 164 Salmonella strains representing all the subspecies of Salmonella enterica were detected while over 50 non- Salmonella strains were not detected. The detection limit of the assay was two colony forming units (cfu) per PCR reaction when a pure culture of S. typhimurium was used. Six protocols for the isolation of PCR-amplifiable DNA were evaluated using chicken carcass rinses, ground beef, ground pork and raw milk contaminated with Salmonella. Of the six DNA isolation protocols, a modified sample preparation protocol using the EnviroAmp kit was chosen for subsequent studies because it was reliable, easy to use and efficient for the isolation of PCR-amplifiable DNA from foods. A detection limit of 3–7 cfu per PCR reaction was obtained using food samples that were pre-enriched overnight and then inoculated with Salmonella. The detection limit was below 3 cfu/25 g or 25 ml when foods inoculated with Salmonella were pre-enriched overnight. Naturally contaminated foods (50 chicken carcass rinses and 60 raw milk samples) were examined using both the fluorogenic 5′ nuclease assay and a modified semi-solid rappaport vassiliadis (MSRV) culture method. Thirty four of the 110 samples tested were Salmonella-positive and 74 were Salmonella-negative by both the 5′ nuclease assay and the MSRV method. Two samples were Salmonella-positive by the 5′ nuclease assay, but negative by the MSRV method. The correlation between the 5′ nuclease assay and the MSRV method was over 98%.