Quantification of Leishmania infantum parasites in tissue biopsies by real-time polymerase chain reaction and polymerase chain reaction-enzyme-linked immunosorbent assay

Most of the experimental studies of Leishmania spp. infection require the determination of the parasite load in different tissues. Quantification of parasites by microscopy is not very sensitive and is time consuming, whereas culture microtitrations remain laborious and can be jeopardized by microbial contamination. The aim of this study was to quantify Leishmania infantum parasites by real-time polymerase chain reaction (PCR) using specific DNA TaqMan probes and to compare the efficacy of detection of this technique with a PCR-enzyme-linked immunosorbent assay (ELISA). For this purpose, spleen and liver samples from L. infantum-infected mice were collected during a 3-mo longitudinal study and analyzed by both methods. PCR-ELISA failed to quantify Leishmania spp. DNA in samples with very low or very high numbers of parasites. Real-time PCR was more sensitive than PCR-ELISA, detecting down to a single parasite, and enabled the parasite quantification over a wide, 5-log range. In summary, this study developed a method for absolute quantification of L. infantum parasites in infected organs using real-time TaqMan PCR.     

Quantification of airborne Aspergillus fumigatus spores in rabbit houses by real-time polymerase chain reaction

To accurately quantify airborne Aspergillus fumigatus (A. fumigatus) spores in rabbit houses, the real-time polymerase chain reaction (real-time PCR) and culture-based counting method (CCM) were employed to determine the airborne A. fumigatus spore concentrations. The results showed that, of the three rabbit houses (A, B, and C), the average concentrations of airborne A. fumigatus spores determined by real-time PCR were 3.0 × 10³, 3.3 × 10³, and 1.5 × 10³ spores/m³ air, respectively, while those determined by CCM were 2.5 × 10², 2.8 × 10², and 1.1 × 10² colony-forming unit/m³ air (CFU/m³ air), respectively, i.e., the former concentration was 12–14 times higher than the latter one. Therefore, the conventional CCM underestimated the concentrations of airborne fungal spores, and it is insufficient to determine the microbial aerosol concentration and evaluate the health risk only using CCM.     

Quantification of serum HBXAP DNA in lung cancer patients by quantitative fluorescent polymerase chain reaction

Hepatitis B virus x associated protein (HBXAP), as a subunit of chromatin remodeling and spacing factor, plays a critical role in cancer development through gene amplification. In this study, we aimed to quantify the levels of serum HBXAP DNA, to analyze and compare its diagnostic value with existing clinical parameters in lung cancer, and to potentially provide a novel tumor marker for lung cancer. Serum HBXAP DNA from 65 lung cancer patients and 20 healthy controls was quantified using real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) analysis. The data were analyzed by statistical software SPSS 13.0. We found that serum HBXAP DNA levels in lung cancer patients were higher compared to healthy controls (u = 219.0, p = 0.001) and were closely associated with TNM stage and lymph node metastasis (p = 0.015 and p = 0.016, respectively). However, serum HBXAP DNA levels were not associated with patient age, gender, smoking status, histological type, or tumor size (p > 0.05). We identified a sensitivity of 61.9 % and a specificity of 93.7 % for the ability of HBXAP DNA levels to detect lung cancer at a cutoff value of 1,557.6 copies/μl. The sensitivity for existing lung-tumor markers, such as squamous cell carcinoma antigen, cytokeratin fragment 21-1, and neuron specific enolase, was increased from 35.7 %, 53.5 %, and 56.0 % to 75.0 %, 86.0 %, and 80.0 %, respectively, by inclusion of serum HBXAP DNA. Taken together, quantification of serum HBXAP DNA by FQ-PCR could potentially serve as a novel complementary tool for the clinical screening and detection of lung cancer.     

Quantification of polymerase chain reaction products by affinity-based hybrid collection.

We have used oligonucleotides modified with biotin in the 5'-end as primers in the polymerase chain reaction (PCR)-amplification. This results in the synthesis of 5'-biotinylated DNA molecules, which are detected by hybridization to a labelled probe in solution. The formed hybrids are collected on an avidin-matrix by mediation of the biotin residue of the target molecules. The affinity-based hybrid collection method is quantitative and makes it possible to measure the amount of DNA produced in the PCR-amplification. At low concentrations of template the efficiency of the process is close to 100%, making it possible to detect the presence of a few molecules of target DNA in 25 cycles. With high template concentrations the efficiency of the process is low.     

Quantitative analysis of total mitochondrial DNA: competitive polymerase chain reaction versus real-time polymerase chain reaction

An efficient and effective method for quantification of small amounts of nucleic acids contained within a sample specimen would be an important diagnostic tool for determining the content of mitochondrial DNA (mtDNA) in situations where the depletion thereof may be a contributing factor to the exhibited pathology phenotype. This study compares two quantification assays for calculating the total mtDNA molecule number per nanogram of total genomic DNA isolated from human blood, through the amplification of a 613-bp region on the mtDNA molecule. In one case, the mtDNA copy number was calculated by standard competitive polymerase chain reaction (PCR) technique that involves co-amplification of target DNA with various dilutions of a nonhomologous internal competitor that has the same primer binding sites as the target sequence, and subsequent determination of an equivalence point of target and competitor concentrations. In the second method, the calculation of copy number involved extrapolation from the fluorescence versus copy number standard curve generated by real-time PCR using various dilutions of the target amplicon sequence. While the mtDNA copy number was comparable using the two methods (4.92 +/- 1.01 x 10(4) molecules/ng total genomic DNA using competitive PCR vs 4.90 +/- 0.84 x 10(4) molecules/ng total genomic DNA using real-time PCR), both inter- and intraexperimental variance were significantly lower using the real-time PCR analysis. On the basis of reproducibility, assay complexity, and overall efficiency, including the time requirement and number of PCR reactions necessary for the analysis of a single sample, we recommend the real-time PCR quantification method described here, as its versatility and effectiveness will undoubtedly be of great use in various kinds of research related to mitochondrial DNA damage- and depletion-associated disorders.     

Novel putative protein tyrosine phosphatases identified by the polymerase chain reaction

Protein tyrosine phosphatases (PTPases) are a family of enzymes that specifically dephosphorylate phosphotyrosyl residues in selected protein substrates. To more fully understand the regulatory role of protein tyrosine phosphorylation and dephosphorylation in cellular signal transduction, characterization of PTPases is essential. Using the polymerase chain reaction and degenerate oligonucleotide primers corresponding to conserved amino acid sequences within the catalytic domain of PTPases, we have identified 11 PTPase-related human liver cDNA sequences. Five of these have not been described previously. These results indicate that, like protein tyrosine kinases, PTPases may also comprise a gene family with a large number of members.     

Rapid titer determination of baculovirus by quantitative real-time polymerase chain reaction

Titer determination is a prerequisite for the study of viruses. However, the current available methods are tedious and time-consuming. To improve the efficiency of titer determination, we have developed a rapid and simple method for the routine detection of baculovirus titers using a quantitative real-time PCR. This method is based on the amplification of approximately 150-bp fragments located in the coding regions of selected genes. The PCR was found to be quantitative in a range of 10(3) to 10(9) virus particles per 200 microL of supernatant, and the results were closely correlated with titers detected from 50% tissue culture infectious doses (TCID(50)) of baculovirus. This quantitative real-time PCR requires only 30 min to perform, and the entire titer determination can be accomplished within 1 h without the need for cell seeding or further virus dilution and infection. Because this technology is easy to operate, generates data with high precision, and most importantly is very quick, it will certainly be broadly applied for titer determination of baculoviruses in the future.     

Quantitation of Bacillus clausii in biological samples by real-time polymerase chain reaction

A real-time PCR assay targeting the highly specific erm34 sequence of Bacillus clausii DNA was developed and optimized. The quantitative assay showed a sensitivity level of 10(2) CFU/microl of sample. The method may represent a useful tool for monitoring the role of B. clausii as probiotic in vivo.     

Detection of Streptococcus anginosus from saliva by real-time polymerase chain reaction

AIMS: The purpose of the present investigation was to assess the salivary levels of Streptococcus anginosus in periodontitis patients. METHODS AND RESULTS: The salivary levels of Strep. anginosus were assessed using real-time polymerase chain reaction (PCR). Streptococcus anginosus was detected in 28 of 37 (75.6%) of periodontitis patients and in three of the 20 (15%) healthy subjects. The mean values for bleeding on probing and probing depth in positive patients were statistically higher than those in negative patients. A significant decrease in Strep. anginosus levels was observed after periodontal treatment. CONCLUSIONS: Although the levels of Strep. anginosus are extremely low, they may reflect the status of periodontal health. SIGNIFICANCE AND IMPACT OF THE STUDY: Real-time PCR is a useful method for obtaining the relative quantities of Strep. anginosus from saliva samples and for monitoring the effect of therapy.     

Quantification of Escherichia coli by kinetic 5'-nuclease polymerase chain reaction (real-time PCR) oriented to sfmD gene

AIMS: A kinetic 5'-nuclease polymerase chain reaction (real-time PCR) for the quantification of Escherichia coli was developed. METHODS AND RESULTS: Specific primers and a fluorogenic probe oriented to sfmD gene, encoding a putative outer membrane export usher protein, were designed. The PCR system was highly specific and sensitive for E. coli, as determined with 37 non-E. coli strains (exclusivity, 100%) and 24 E. coli strains (inclusivity, 100%). When used in real-time PCR, linear calibration lines were obtained in the range from 10(2) to 10(8) CFU ml(-1) for three E. coli strains. Salmonella Enteritidis (10(6) CFU ml(-1)) or Citrobacter freundii (10(6) CFU ml(1)) had no effect on quantification of E. coli by the method. CONCLUSIONS: The developed real-time PCR is suitable for rapid quantification of E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: In connection to an appropriate sample preparation technique, the method is suitable for food safety and technological hygiene applications.     

BK virus DNA detection by real-time polymerase chain reaction in clinical specimens

The BK polyomavirus (BKV) is widespread in the general population. In transplant recipients, the patients' weakened immune response may encourage reactivation of latent infection, leading to BKV-related diseases. Rapid and quantitative detection might help to delineate viral reactivation patterns and could thus play an important role in their clinical management. In our study we developed an "in-house" quantitative real-time PCR to detect BKV DNA. The effectiveness of this assay was evaluated by a retrospective analysis of 118 plasma specimens from 22 bone marrow transplant (BMT) recipients and 107 samples from immunocompetent subjects. Eight (36.3%) of the 22 bone marrow transplant recipients tested positive for BKV. The viral load varied from specimen to specimen (10 to 10(5) copies/ml). BKV related disease like hemorrhagic cystitis (HC) was diagnosed in three patients. Specimens from the control group all tested negative. Our results showed the high sensitivity of the real-time PCR, allowing accurate and reproducible measuring of the viral load in order to identify patients at risk for BKV-related diseases. With due caution in interpreting threshold values, the real-time PCR could provide a rapid, sensitive and specific tool for detecting BKV and distinguishing latent and active infection.     

Detection of infectious dengue virus by selective real-time quantitative polymerase chain reaction

Dear Editor,The dengue virus(DENV)is a single-stranded positivesense RNA virus that belongs to the family Flaviviridae(Gubler,2002),and has four serotypes,DENV1–DENV4,which are transmitted via Aedes aegypti and Aedes albopictus(Rodriguez-Roche and Gould,2013).It has been reported that more than 50 million dengue infections occur each year(Guzman et al.,2010),and a serious outbreak occurred in the Southern Provinces of