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.     

Matrix approach to the simultaneous detection of multiple potato pathogens by real‐time PCR

Aim

Create a method for highly sensitive, selective, rapid and easy‐to‐use detection and identification of economically significant potato pathogens, including viruses, bacteria and oomycetes, be it single pathogen, or a range of various pathogens occurring simultaneously.

Methods and Results

Test‐systems for real‐time PCR, operating in the unified amplification regime, have been developed for Phytophthora infestans, Pectobacterium atrosepticum, Dickeya dianthicola, Dickeya solani, Ralstonia solanacearum, Pectobacterium carotovorum, Clavibacter michiganensis subsp. sepedonicus, potato viruses Y (ordinary and necrotic forms as well as indiscriminative test system, detecting all forms), A, X, S, M, potato leaf roll virus, potato mop top virus and potato spindle tuber viroid. The test‐systems (including polymerase and revertase) were immobilized and lyophilized in miniature microreactors (1·2 μl) on silicon DNA/RNA microarrays (micromatrices) to be used with a mobile AriaDNA^® amplifier.

Conclusions

Preloaded 30‐reaction micromatrices having shelf life of 3 and 6 months (for RNA‐ and DNA‐based pathogens, respectively) at room temperature with no special conditions were successfully tested on both reference and field samples in comparison with traditional ELISA and microbiological methods, showing perfect performance and sensitivity (1 pg).

Significance and Impact of the Study

The accurate, rapid and user‐friendly diagnostic system in a micromatrix format may significantly contribute to pathogen screening and phytopathological studies.     

A comparison of DNA methylation specific droplet digital PCR (ddPCR) and real time qPCR with flow cytometry in characterizing human T cells in peripheral blood

Quantitating the copy number of demethylated CpG promoter sites of the CD3Z gene can be used to estimate the numbers and proportions of T cells in human blood and tissue. Quantitative methylation specific PCR (qPCR) is useful for studying T cells but requires extensive calibration and is imprecise at low copy numbers. Here we compared the performance of a new digital PCR platform (droplet digital PCR or ddPCR) to qPCR using bisulfite converted DNA from 157 blood specimens obtained from ambulatory care controls and patients with primary glioma. We compared both ddPCR and qPCR with conventional flow cytometry (FACS) evaluation of CD3 positive T cells. Repeated measures on the same blood sample revealed ddPCR to be less variable than qPCR. Both qPCR and ddPCR correlated significantly with FACS evaluation of peripheral blood CD3 counts and CD3/total leukocyte values. However, statistical measures of agreement showed that linear concordance was stronger for ddPCR than for qPCR and the absolute values were closer to FACS for ddPCR. Both qPCR and ddPCR could distinguish clinically significant differences in T cell proportions and performed similarly to FACS. Given the higher precision, greater accuracy, and technical simplicity of ddPCR, this approach appears to be a superior DNA methylation based method than conventional qPCR for the assessment of T cells.     

A comparison of DNA methylation specific droplet digital PCR (ddPCR) and real time qPCR with flow cytometry in characterizing human T cells in peripheral blood

Quantitating the copy number of demethylated CpG promoter sites of the CD3Z gene can be used to estimate the numbers and proportions of T cells in human blood and tissue. Quantitative methylation specific PCR (qPCR) is useful for studying T cells but requires extensive calibration and is imprecise at low copy numbers. Here we compared the performance of a new digital PCR platform (droplet digital PCR or ddPCR) to qPCR using bisulfite converted DNA from 157 blood specimens obtained from ambulatory care controls and patients with primary glioma. We compared both ddPCR and qPCR with conventional flow cytometry (FACS) evaluation of CD3 positive T cells. Repeated measures on the same blood sample revealed ddPCR to be less variable than qPCR. Both qPCR and ddPCR correlated significantly with FACS evaluation of peripheral blood CD3 counts and CD3/total leukocyte values. However, statistical measures of agreement showed that linear concordance was stronger for ddPCR than for qPCR and the absolute values were closer to FACS for ddPCR. Both qPCR and ddPCR could distinguish clinically significant differences in T cell proportions and performed similarly to FACS. Given the higher precision, greater accuracy, and technical simplicity of ddPCR, this approach appears to be a superior DNA methylation based method than conventional qPCR for the assessment of T cells.     

Quantification of Lawsonia intracellularis in porcine faeces by real‐time PCR

Aim: To develop and to validate a method for the quantification of Lawsonia intracellularis in porcine faeces by real‐time PCR. Methods and Results: A real‐time PCR including a calibrator based on plasmid DNA for quantification by means of ΔΔCt method was evaluated. The parameters specificity, detection limit, quantification limit, linearity, range, repeatability, precision and recovery were validated. The detection limit of the agent was 1 copy per reaction, and quantification was reliable between 10¹ and 10⁷ copies per μl reaction volume. The linearity calculated by logistic regression revealed a slope of ?3·329 reflecting an efficiency of 99·7% for the assay. Moreover, it was shown that storage of samples and repetition of tests including DNA isolation by same or other investigators did not influence the outcome. Conclusion: The quantification method described herein revealed consistent results for the quantitation of L. intracellularis in porcine faeces samples. Significance and Impact of the Study: In contrast to common PCR in combination with gel electrophoresis, this validated quantification method based on real‐time PCR enhances a reliable quantification and is even more sensitive.     

Development and comparison of SYBR Green quantitative real‐time PCR assays for detection and enumeration of sulfate‐reducing bacteria in stored swine manure

Aims: To develop and evaluate primer sets targeted to the dissimilatory sulfite reductase gene (dsrA) for use in quantitative real‐time PCR detection of sulfate‐reducing bacteria (SRB) in stored swine manure. Methods and Results: Degenerate primer sets were developed to detect SRB in stored swine manure. These were compared with a previously reported primer set, DSR1F+ and DSR‐R, for their coverage and ability to detect SRB communities in stored swine manure. Sequenced clones were most similar to Desulfovibrio sp. and Desulfobulbus sp., and these SRB populations differed within different manure ecosystems. Sulfur content of swine diets was shown to affect the population of Desulfobulbus‐like Group 1 SRB in manure. Conclusions: The newly developed assays were able to enumerate and discern different groups of SRB, and suggest a richly diverse and as yet undescribed population of SRB in swine manure. Significance and Impact of the Study: The PCR assays described here provide improved and efficient molecular tools for quantitative detection of SRB populations. This is the first study to show population shifts of SRB in swine manure, which are a result of either the effects of swine diets or the maturity of the manure ecosystem.     

Rapid Detection of Colletotrichum lagenarium,Causal Agent of Anthracnose of Cucurbitaceous Crops,by PCR and Real‐time PCR

Rapid and accurate polymerase chain reaction (PCR) and real‐time PCR methods were developed for the detection of Colletotrichum lagenarium, the causal agent of anthracnose, in tissues of squash (Cucurbita moschata), watermelon (Citrullus lanatus), cucumber (Cucumis sativus) and muskmelon (Cucumis melo). PCR assays amplified different internal transcribed spacer sequences from C. lagenarium, so effectively detected this pathogen in infected tissues. PCR analysis with the primer co‐m‐337F1/R1 was able to differentiate C. lagenarium from other fungal pathogens, including Colletotrichum spp., Fusarium spp., Alternaria spp. and Didymella spp. An optimized real‐time PCR assay was developed to detect and monitor C. lagenarium in both infected plant tissues and soil samples. The sensitivity of real‐time PCR can detect down to 1 pg of DNA. Thus, PCR‐based analysis is a useful technique for rapid detection and diagnosis of C. lagenarium in infected plants or infested soils.     

A real‐time PCR assay for improved rapid,specific detection of Cryphonectria parasitica

Cryphonectria parasitica, an ascomycete fungus, is the causal agent of chestnut blight. This highly destructive disease of chestnut trees causes significant losses, and is therefore a regulated pathogen in Europe. Existing methods for the detection of C. parasitica include morphological identification following culturing, or PCR; however, these are time‐consuming resulting in delays to diagnosis. To allow improved detection, a new specific real‐time PCR assay was designed to detect C. parasitica directly from plant material and fungal cultures, and was validated according to the European Plant Protection Organisation (EPPO) standard PM 7/98. The analytical specificity of the assay was tested extensively using a panel of species taxonomically closely related to Cryphonectria, fungal species associated with the hosts and healthy plant material. The assay was found to be specific to C. parasitica, whilst the analytical sensitivity of the assay was established as 2 pg µL^?1 of DNA. Comparative testing of 63 samples of naturally infected plant material by the newly developed assay and traditional morphological diagnosis demonstrated an increased diagnostic sensitivity when using the real‐time PCR assay. Furthermore the assay is able to detect both virulent and hypovirulent strains of C. parasitica. Therefore the new real‐time PCR assay can be used to provide reliable, rapid, specific detection of C. parasitica to prevent the accidental movement of the disease and to monitor its spread.     

Evaluation of Legionella pneumophila contamination in Italian hotel water systems by quantitative real‐time PCR and culture methods

Aims: This study was designed to define the extent of water contamination by Legionella pneumophila of certain Italian hotels and to compare quantitative real‐time PCR with the conventional culture method. Methods and Results: Nineteen Italian hotels of different sizes were investigated. In each hotel three hot water samples (boiler, room showers, recycling) and one cold water sample (inlet) were collected. Physico‐chemical parameters were also analysed. Legionella pneumophila was detected in 42% and 74% of the hotels investigated by the culture method and by real‐time PCR, respectively. In 21% of samples analysed by the culture method, a concentration of >10⁴ CFU l^?1 was found, and Leg. pneumophila serogroup 1 was isolated from 10·5% of the hotels. The presence of Leg. pneumophila was significantly influenced by water sample temperature, while no association with water hardness or residual‐free chlorine was found. Conclusions: This study showed a high percentage of buildings colonized by Leg. pneumophila. Moreover, real‐time PCR proved to be sensitive enough to detect lower levels of contamination than the culture method. Significance and Impact of the Study: This study indicates that the Italian hotels represent a possible source of risk for Legionnaires’ disease and confirms the sensitivity of the molecular method. To our knowledge, this is the first report to demonstrate Legionella contamination in Italian hotels using real‐time PCR and culture methods.     

Development and evaluation of real‐time PCR assays for bloodmeal identification in Culicoides midges

Culicoides (Diptera: Ceratopogonidae) midges are the biological vectors of a number of arboviruses of veterinary importance. However, knowledge relating to the basic biology of some species, including their host‐feeding preferences, is limited. Identification of host‐feeding preferences in haematophagous insects can help to elucidate the transmission dynamics of the arboviruses they may transmit. In this study, a series of semi‐quantitative real‐time polymerase chain reaction (qPCR) assays to identify the vertebrate host sources of bloodmeals of Culicoides midges was developed. Two pan‐reactive species group and seven species‐specific qPCR assays were developed and evaluated. The assays are quick to perform and less expensive than nucleic acid sequencing of bloodmeals. Using these assays, it was possible to rapidly test nearly 700 blood‐fed midges of various species from several geographic locations in Australia.     

Multiplex real‐time PCR assays for detection of four seedborne spinach pathogens

Aims

To develop multiplex TaqMan real‐time PCR assays for detection of spinach seedborne pathogens that cause economically important diseases on spinach.

Methods and Results

Primers and probes were designed from conserved sequences of the internal transcribed spacer (for Peronospora farinosa f. sp. spinaciae and Stemphylium botryosum), the intergenic spacer (for Verticillium dahliae) and the elongation factor 1 alpha (for Cladosporium variabile) regions of DNA. The TaqMan assays were tested on DNA extracted from numerous isolates of the four target pathogens, as well as a wide range of nontarget, related fungi or oomycetes and numerous saprophytes commonly found on spinach seed. Multiplex real‐time PCR assays were evaluated by detecting two or three target pathogens simultaneously. Singular and multiplex real‐time PCR assays were also applied to DNA extracted from bulked seed and single spinach seed.

Conclusions

The real‐time PCR assays were species‐specific and sensitive. Singular or multiplex real‐time PCR assays could detect target pathogens from both bulked seed samples as well as single spinach seed.

Significance and Impact of the Study

The freeze‐blotter assay that is currently routinely used in the spinach seed industry to detect and quantify three fungal seedborne pathogens of spinach (C. variabile, S. botryosum and V. dahliae) is quite laborious and takes several weeks to process. The real‐time PCR assays developed in this study are more sensitive and can be completed in a single day. As the assays can be applied easily for routine seed inspections, these tools could be very useful to the spinach seed industry.     

Evaluation of real‐time PCR methods for quantification of Acanthamoeba in anthropogenic water and biofilms

Aims: To assess two real‐time PCR methods (the Riviere and Qvarnstrom assays) for environmental Acanthamoeba. Methods and Results: DNA extracted from Acanthamoeba castellanii taken from water and biofilms of cooling towers was analysed by the Riviere and Qvarnstrom assays. To quantify environmental Acanthamoeba, the calibration curves (DNA quantity vs cell number) were constructed with samples spiked with A. castellanii. The calibration curves for both quantitative PCR assays showed low variation (coefficient of variation of C_t≤ 5·7%) and high linearity (R² ≥ 0·99) over six orders of magnitudes with detection limit of three cells per water sample. DNA quantity determined by Qvarnstrom assay was equivalent between trophozoites and cysts (P = 0·49), whereas a significant difference was observed with Riviere assay (P < 0·0001). Riviere assay failed to detect Acanthamoeba in 21% (15/71) of the environmental samples which were positively detected by Qvarnstrom assay, while one sample (1·4%) was shown positive by Riviere assay but negative by Qvarnstrom assay. Moreover, Acanthamoeba counts by Qvarnstrom assay were greater than those by Riviere assay (P < 0·0001). Conclusions: Qvarnstrom assay performs better than Riviere assay for detection and quantification of Acanthamoeba in anthropogenic water and biofilms. Significance and Impact of the Study: Qvarnstrom assay may significantly contribute to a better knowledge about the distribution and abundance of Acanthamoeba in environments.     

Hepatitis E virus‐based evaluation of a virion concentration method and detection of enteric viruses in environmental samples by multiplex nested RT‐PCR

Aims: The prevalence of enteric viruses in drinking and river water samples collected from Pune, India was assessed. During an outbreak of HEV in a small town near pune, water samples were screened for enteric viruses. Methods and Results: The water samples were subjected to adsorption–elution‐based virus concentration protocol followed by multiplex nested PCR. Among 64 Mutha river samples, 49 (76·56%) were positive for Hepatitis A Virus, 36 (56·25%) were positive for Rotavirus, 33 (51·56%) were positive for Enterovirus and 16 (25%) were positive for Hepatitis E Virus RNA. Only enterovirus RNA was detected in 2/662 (0·3%) drinking water samples, and the samples from the city’s water reservoir tested negative for all four viruses. HEV RNA was detected in three out of four river water samples during HEV outbreak and partial sequences from patients and water sample were identical. Conclusions: The study suggests absence of enteric viruses both in the source and in the purified water samples from Pune city, not allowing evaluation of the purification system and documents high prevalence of enteric viruses in river water, posing threat to the community. Significance and Impact of the Study: The rapid, sensitive and relatively inexpensive protocol developed for virological evaluation of water seems extremely useful and should be adapted for evaluating viral contamination of water for human consumption. This will lead to development of adequate control measures thereby reducing disease burden because of enteric viruses.