Evaluation of two TaqMan PCR assays for the detection of HIV-1 proviral DNA in blood samples

This study compared two published TaqMan PCR assays targeting different regions of the HIV-1 genome for detection of HIV-1 proviral DNA. The gag specific PCR demonstrated a lower sensitivity than the assay targeting the LTR region. The LTR assay is a highly reproducible and specific technique for HIV-1 proviral DNA detection.     

A new set of PCR assays for the identification of multiple human adenovirus species in environmental samples

Aims:  To assess human adenovirus (HAdV) diversity in environmental samples based on sequence comparisons of hexon gene fragments amplified using newly designed HAdV-specific polymerase chain reaction (PCR) assays.
Methods and Results:  Six PCR primer sets were designed based on 56 aligned hexon sequences from NCBI GenBank to amplify different hexon gene sections (241–349 bp) of the six HAdV species. The amplified hexon genes from wastewater samples were cloned, sequenced, and compared with those in publicly accessible databases (i.e. NCBI GenBank) by using the B last program. A total of 46 analysed positive clones were affiliated to five HAdV serotypes, i.e. 1, 2, 12, 31 and 41. Similarities between the cloned and database hexon sequences ranged from 95·9 to 100% (with an average of 98·1 ± 1·0%).
Conclusion:  The designed primers showed higher amplification efficiencies when compared with the existing assays. Using the new assays, HAdV species A, C, and F (serotypes 1, 2, 12, 31 and 41 in particular) were identified in the studied municipal wastewater.
Significance and Impact of the Study:  The six PCR primer sets developed in this study can be used to efficiently amplify hexon gene fragments in HAdV. Multiple HAdV serotypes identified in the municipal wastewater provide new information about HAdV diversity in environmental samples.     

Comparison of the host specificities of two bacteroidales quantitative PCR assays used for tracking human fecal contamination

The sewage-associated real-time quantitative PCR (qPCR) assays BacHum and HF183 SYBR were compared for specificity against local fecal sources. Both assays were equally sensitive to sewage, but BacHum showed substantially more false-positive results for cat, dog, gull, and raccoon feces.     

Comparison of different PCR methods for detection of Brucella spp. in human blood samples

For detection of Brucella species by PCR four DNA extraction methods and four targets were compared using pure culture of Brucella melitensis and the best conditions were applied in clinical samples. It was found that the MagNA Pure LC method was the most efficient and sensitive method showing a positive PCR reaction with DNA extracted from as low as 25 and 100 CFU suspended in one ml blood and one ml water, respectively. Detection of Brucella spp. by conventional PCR was investigated using four different targets. The results indicated that The B4-B5 amplification method was the most sensitive one as it could amplify DNA extracted from as a low as 25 and 100 CFU/ml suspended in one ml water and blood, respectively. Furthermore real-time PCR was able to detect Brucella using DNA extracted from as low as 50 CFU/ml blood and 15 CFU/ml water, respectively. The best and optimum detection conditions were applied to the clinical samples. Evaluation of conventional PCR assays on blood specimens confirmed 72% of the results obtained by conventional blood culture methods with a specificity of 95%, while serum samples had a sensitivity of 54% and specificity of 100%. Real-time PCR was generally found to be more sensitive and specific for detecting Brucella spp. in blood and serum samples compared to conventional PCR. The real-time PCR done on blood specimens confirmed 77.5% of the results obtained by conventional blood culture methods with specificity of 100%, while 60% of serum samples were found to be positive with specificity of 100%. These results suggest that serum and blood analysis by conventional and real time PCR is a convenient and safe method for rapid and accurate diagnosis of brucellosis.     

Comparison of three different PCR methods for detection of Brucella spp in human blood samples

In the diagnosis of human brucellosis, PCR could be a more sensitive technique than blood cultures and more specific than conventional serological tests. We compared three different PCR methods for the detection of Brucella spp. and we studied whether human genomic DNA affect the sensitivity of three primer pairs for the detection of Brucella DNA in a peripheral-blood PCR assay. These three pairs of primers amplified three different fragments included in: (i). a gene encoding a 31-kDa Brucella abortus antigen (primers B4/B5), (ii). a sequence 16S rRNA of B. abortus (primers F4/R2), and (iii). a gene encoding an outer membrane protein (omp-2) (primers JPF/JPR). The three primers assayed showed a difference in sensitivity for detecting purified Brucella DNA, ranging between 8 fg and 20 pg. However, the sensitivity of the primers F4/R2 and B4/B5 was affected by the presence of human DNA while the primers JPF/JPR were not. Therefore, although the sensitivity of PCR using primers F4/R2 is affected by human DNA, they are still the most sensitive and they could provide a useful tool for the diagnosis of human brucellosis.     

Development of stable isotope dilution assays for ochratoxin A in blood samples

Two new stable isotope dilution assays were developed for the quantification of ochratoxin A in human blood samples for exposure studies. The methods based on two different sample extraction and cleanup procedures including liquid–liquid extraction with following immunoaffinity chromatography (IA) as well as a dispersive solid-phase extraction (DSPE) method. For detection, LC–MS/MS was applied. For the first time, exact quantitation of the reference compound ochratoxin A was performed by quantitative NMR spectroscopy (qNMR). Additionally, a comparison of different blood-drawing procedures revealed no differences for heparin plasma and serum whereas citrate plasma gave significantly lower results for the mycotoxin. Limits of detection (LOD: 0.02 ng/g (IA) vs 0.03 ng/g (DSPE)), limits of quantification (LOQ: 0.07 ng/g (IA) vs 0.08 ng/g (DSPE)), relative recovery (?94%), precision, and linearity indicated excellent performance of the developed methods.     

Use of PCR analysis for detecting low levels of bacteria and mold contamination in pharmaceutical samples

PCR assays were developed and compared to standard methods for quality evaluation of pharmaceutical raw materials and finished products with low levels of microbial contamination. Samples were artificially contaminated with less than 10 CFU of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Aspergillus niger. Bacterial DNA was extracted from each enrichment broth by mild lysis in Tris-EDTA-Tween 20 buffer containing proteinase K while mold DNA was extracted by boiling samples in Tris-EDTA-SDS buffer for 1 h. A 10-microl aliquot of extracted DNA was added to Ready-To-Go PCR beads and specific primers for E. coli, S. aureus, and P. aeruginosa. However, 50-microl aliquots of extracted mold DNA were used for amplification of specific A. niger DNA sequences. Standard methods required 6-8 days while PCR detection of all microorganisms was completed within 27 h. Low levels of microbial contamination were detected in all raw materials and products using PCR assays. Rapid quality evaluation of pharmaceutical samples resulted in optimization of product manufacturing, quality control, and release of finished products.     

Evaluating genomic DNA extraction methods from human whole blood using endpoint and real-time PCR assays

The extraction of genomic DNA is the crucial first step in large-scale epidemiological studies. Though there are many popular DNA isolation methods from human whole blood, only a few reports have compared their efficiencies using both end-point and real-time PCR assays. Genomic DNA was extracted from coronary artery disease patients using solution-based conventional protocols such as the phenol–chloroform/proteinase-K method and a non-phenolic non-enzymatic Rapid-Method, which were evaluated and compared vis-a-vis a commercially available silica column-based Blood DNA isolation kit. The appropriate method for efficiently extracting relatively pure DNA was assessed based on the total DNA yield, concentration, purity ratios (A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀), spectral profile and agarose gel electrophoresis analysis. The quality of the isolated DNA was further analysed for PCR inhibition using a murine specific ATP1A3 qPCR assay and mtDNA/Y-chromosome ratio determination assay. The suitability of the extracted DNA for downstream applications such as end-point SNP genotyping, was tested using PCR-RFLP analysis of the AGTR1-1166A>C variant, a mirSNP having pharmacogenetic relevance in cardiovascular diseases. Compared to the traditional phenol–chloroform/proteinase-K method, our results indicated the Rapid-Method to be a more suitable protocol for genomic DNA extraction from human whole blood in terms of DNA quantity, quality, safety, processing time and cost. The Rapid-Method, which is based on a simple salting-out procedure, is not only safe and cost-effective, but also has the added advantage of being scaled up to process variable sample volumes, thus enabling it to be applied in large-scale epidemiological studies.     

Detection of Legionella pneumophila in water samples by species-specific real-time and nested PCR assays

AIMS: Legionella pneumophila is a contaminant of man-made water systems, including potable water, cooling towers, water systems of large buildings, etc. It is the most common causative agent of legionellosis, a respiratory infection, which may give rise to restricted outbreaks. To survey environmental water samples from hospitals and private habitations in Bologna, we developed a species-specific nested and a TaqMan real-time PCR for the detection of L. pneumophila. We compared the two assays and both to cultural isolation. METHODS AND RESULTS: The targeted gene was macrophage infectivity potentiator (mip), conserved in L. pneumophila, and divergent in other legionellae. One assay was based on a nested PCR and the other on a TaqMan real-time PCR protocol. Their sensitivities were 14 % or 5% higher than that of cultural isolation respectively. The detection limits were 1-2 genome equivalents per 50-microl reaction. Specificity was assessed using DNA from nine target and 20 nontarget organisms. CONCLUSIONS: When applied to water samples, both assays detected L. pneumophila at 80% or higher frequency. SIGNIFICANCE AND IMPACT OF THE STUDY: The species-specific molecular diagnosis of L. pneumophila by means of nested PCR does not require a specific instrumentation, exhibits a high sensitivity, and is advantageous over the cultural isolation and real-time PCR detection. It allows to quickly monitor water samples for the risk assessment of environmental contaminations.     

Quantitative PCR assays to survey the bovine adenovirus levels in environmental samples

Aims: Previous studies suggested bovine adenoviruses (BAdVs) could be used as cattle faecal indicators. The main aim of this study was to survey the levels of BAdV in environmental samples using quantitative polymerase chain reaction (qPCR). Methods and Results: Two qPCR assays were developed to identify and quantify BAdVs in environmental samples. BAdVs were detected in all dairy manure, and in most cases, the sample concentrations were around 10³–10⁴ copies ml^?1. Farm tile drainage samples were also detected, but the concentrations were about 1–3 log₁₀ lower than the BAdV concentrations in the manure samples. The genome equivalent copy (GEC) levels of BAdV and the phage forming unit (PFU) levels of somatic phage in manure samples were comparable. Four of twenty individual cattle faeces were positive with concentrations similar to that found in the manure samples. Sequencing results confirmed the presence of BAdV in the environmental samples, and phylogenetic analysis indicated that BAdV 2 and 4 were the most prevalent serotypes in all the manure samples tested. The qPCR assays developed in this study showed higher sensitivity in detecting BAdV 1 and 2 than the previous published nested assay. Conclusion: The high levels of BAdV in the environmental samples may suggest it could be used for bovine faecal indicator. The significant levels of BAdV in the drainage samples may indicate the potential of surface water pollution by the manure applied to farm fields. Significance and Impact of the Study: This is the first study that reports the quantitative level of BAdV in environmental samples. These results could be useful when it comes to determining whether BAdV could be utilized as a bovine faecal indicator.     

Hypersensitive PCR, ancient human mtDNA, and contamination

When highly efficient polymerase was used with high cycle numbers (50-60), strong amplifications were observed, but negative controls were also unexpectedly amplified in a study of ancient human mtDNA from 2000-year-old skeletons. The results of a series of tests revealed that the hypersensitive polymerase chain reaction (PCR) generated by higher cycles and the presence of contaminant DNA (though at extremely low levels) should be responsible for the amplification of negative controls. We suggest that PCR sensitivity be optimized to take advantage of highly efficient polymerase and at the same time prevent "background DNA" from becoming "contaminant DNA" and obscuring the analysis of authentic ancient DNA. We propose the use of multiple positive controls when amplifying ancient human mtDNA samples to indicate the sensitivity of individual PCR amplifications and to monitor the contamination levels of modern human DNA. This study provides some suggestions as to how to amplify and analyze ancient human mtDNA when unavoidable and extremely tiny amounts of modern human DNA exist.     

Diagnosis of human visceral leishmaniasis by PCR using blood samples spotted on filter paper

The polymerase chain reaction (PCR) is a simple, rapid procedure that has been adapted for the diagnosis of leishmaniasis. In the present study, 85 blood samples and seven bone marrow aspirates from 85 patients with clinical symptoms suggestive of visceral leishmaniasis from the metropolitan region of Belo Horizonte in the Brazilian State of Minas Gerais were screened using molecular and serological techniques. Samples that were negative (N = 12) and positive (N = 19) in parasitological and serological tests were used as controls. Of the 85 samples analyzed by PCR, 61 (71.7%) showed the expected amplification products in agarose gels. However, when the technique was combined with molecular hybridization, 72 samples (83.5%) gave a positive signal on film. Nineteen patients with Leishmania parasites in bone marrow cultures (positive controls) showed PCR hybridization in whole-blood samples, as did the seven bone marrow aspirates positive for Leishmania. None of the negative controls reacted in PCR or in an indirect immunofluorescent assay. These results indicate that PCR could replace the conventional parasitological examination in the diagnosis of leishmaniasis since it provides very satisfactory results with blood samples spotted on filter paper.     

Identification of replication competent murine gammaretroviruses in commonly used prostate cancer cell lines

A newly discovered gammaretrovirus, termed XMRV, was recently reported to be present in the prostate cancer cell line CWR22Rv1. Using a combination of both immunohistochemistry with broadly-reactive murine leukemia virus (MLV) anti-sera and PCR, we determined if additional prostate cancer or other cell lines contain XMRV or MLV-related viruses. Our study included a total of 72 cell lines, which included 58 of the 60 human cancer cell lines used in anticancer drug screens and maintained at the NCI-Frederick (NCI-60). We have identified gammaretroviruses in two additional prostate cancer cell lines: LAPC4 and VCaP, and show that these viruses are replication competent. Viral genome sequencing identified the virus in LAPC4 and VCaP as nearly identical to another known xenotropic MLV, Bxv-1. We also identified a gammaretrovirus in the non-small-cell lung carcinoma cell line EKVX. Prostate cancer cell lines appear to have a propensity for infection with murine gammaretroviruses, and we propose that this may be in part due to cell line establishment by xenograft passage in immunocompromised mice. It is unclear if infection with these viruses is necessary for cell line establishment, or what confounding role they may play in experiments performed with these commonly used lines. Importantly, our results suggest a need for regular screening of cancer cell lines for retroviral "contamination", much like routine mycoplasma testing.     

Evaluation of the performance of selected in-house and commercially available PCR and real-time PCR assays for the detection of Leishmania DNA in canine clinical samples

Protozoa of the genus Leishmania are the causative agents of leishmaniosis. Although the polymerase chain reaction (PCR) has proved very effective in the detection of Leishmania DNA, a standardized method does not exist. In this study we attempt a comparative evaluation between one real time PCR (Method D), two in-house (Methods A and C), and a commercially available PCR assay (Method B) for the detection of Leishmania DNA, in order to support reliable diagnostic investigation of leishmaniosis. This evaluation was performed in regard to relative specificity and sensitivity, minimum detection limit (MDL), repeatability and reproducibility using cultured isolates and clinical samples. All the methods under study produced the expected result with the positive and negative controls. However with regard to clinical samples, Method C showed a statistically significant higher level of positivity. Relative sensitivity and specificity of Methods A, B and D in comparison to C was calculated respectively at 50.7%, 43%, 40%, and 90.8%, 93.4% and 89.5%. The MDL for Methods A-D was defined respectively at 30.7, 5, 3.7, and 5 promastigotes/ml. Repeatability and reproducibility were excellent in all cases with only the exception of Method A regarding reproducibility with a different brand of PCR reagents. The results that were recorded indicate that evaluation of PCR assays before their application for research and clinical diagnosis can provide useful evidence for their reliable application. Within this context the use of internal amplification controls and the confirmation of the specificity of the amplification product is recommended.     

Evaluation of nested PCR assays for the detection of Legionella pneumophila in a wide range of aquatic samples

AIMS: To compare the sensitivities of two nested PCR assays for the detection of Legionella pneumophila to each other and to the plate counting technique (ISO 11731) in a wide range of aquatic samples. METHODS AND RESULTS: The nested PCR assay with the primer set LEG 225-LEG 858 revealed 56% of the 46 analysed aquatic samples as being positive for Legionella spp., while the primer set JFP-JRP yielded 98% positive samples. The detection was confirmed by sequencing the PCR products. These results are considerably higher than the result obtained with the plate counting technique (41%), indicating the higher sensitivity of PCR-based diagnostic methods. As the PCR assay with the LEG 225-LEG 858 primer set resulted in a lower number of positive samples, it is considered not sensitive enough for aquatic samples. Similar results for the respective primer sets were obtained for the detection of the species L. pneumophila, responsible for 90% of all human Legionella infections, in the aquatic samples analysed. Both microbial community analysis by PCR-denaturing gradient gel electrophoresis and the analysis of biotic and abiotic water quality parameters revealed no relation between L. pneumophila-positive and -negative samples and the physico-chemical and bacteriological characteristics of the aquatic samples. CONCLUSIONS: The results show the additional value of the PCR assay with the JFP-JRP primer set compared with the plate counting technique, as well as its applicability in a wide range of aquatic samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows the importance of comparing different primer sets for nested PCR assays for the detection of L. pneumophila in aquatic samples, as well as the lower sensitivity of the widely accepted plate counting technique (ISO 11731).     

Differences in amplification efficiency of standard curves in quantitative real-time PCR assays and consequences for gene quantification in environmental samples

High and comparable efficiency values are the key for reliable quantification of target genes from environmental samples using real-time PCR. Therefore it was the aim of this study to investigate if PCR amplification efficiencies of plasmid DNA used for the calculation of standard curves (i) remain constant along a logarithmic scale of dilutions and (ii) if these values are comparable to those of DNA extracted from environmental samples. It could be shown that comparable efficiency values within the standards cannot be achieved using log scale serial dilutions and a comparison of gene copy numbers from DNA extracted from environmental samples and standard DNA extracted from plasmids is only possible in a very small interval.     

Multicenter evaluation of prototype real‐time PCR assays for Epstein‐Barr virus and cytomegalovirus DNA in whole blood samples from transplant recipients

Quantitative PCR is becoming widespread for diagnosing and monitoring post‐transplantation diseases associated with EBV and CMV. These assays need to be standardized to manage patients in different facilities. Five independent laboratories in Japan compared home‐brew assays and a prototype assay system to establish a standard quantitative procedure for measuring EBV and CMV. Reference standards and a total of 816 (642 EBV and 174 CMV) whole blood samples from post‐transplantation recipients were used for this multicenter evaluation. The prototype reference standard for EBV was compared to a panel of samples, with a theoretical expected value made using EBV‐positive cells containing two virus genome copies per cell. The mean ratio of the reference standard at each site to the standard of the prototype assay was ≤4.15 for EBV among three different sites and ≤3.0 for CMV between two laboratories. The mean of the theoretical expected number of the EBV genome: prototype reference was close to 1.0. The correlation coefficients between the viral copy numbers determined using the prototype assay and those using each home‐brew assay were high (EBV, 0.73–0.83, median = 0.78; CMV, 0.54–0.60, median = 0.57). The dynamics of the EBV and CMV loads in transplant recipients were similar between the assay types. There was an inter‐laboratory difference among the quantification results, indicating that a unified protocol and kit are favorable for standardizing the quantification of EBV and CMV. Such standardization will help to standardize the diagnosis and monitoring of diseases associated with EBV and CMV.