Development of a rotor-gene real-time PCR assay for the detection and quantification of Mycoplasma genitalium

We developed and validated a real-time quantitative polymerase chain reaction (qPCR) assay to determine Mycoplasma genitalium bacterial load in endocervical swabs, based on amplification of the pdhD gene which encodes dihydrolipoamide dehydrogenase, using the Rotor-Gene platform. We first determined the qPCR assay sensitivity, limit of detection, reproducibility and specificity, and then determined the ability of the qPCR assay to quantify M. genitalium in stored endocervical specimens collected from Zimbabwean women participating in clinical research undertaken between 1999 and 2007. The qPCR assay had a detection limit of 300 genome copies/mL and demonstrated low intra- and inter-assay variability. The assay was specific for M. genitalium DNA and did not amplify the DNA from other mycoplasma and ureaplasma species. We quantified M. genitalium in 119 of 1600 endocervical swabs that tested positive for M. genitalium using the commercial Sacace M. genitalium real-time PCR, as well as 156 randomly selected swabs that were negative for M. genitalium by the same assay. The M. genitalium loads ranged between < 300 and 3,240,000 copies/mL. Overall, the qPCR assay demonstrated good range of detection, reproducibility and specificity and can be used for both qualitative and quantitative analyses of M. genitalium in endocervical specimens and potentially other genital specimens.     

非洲猪瘟病毒微滴数字PCR (ddPCR)方法的建立及应用

【目的】非洲猪瘟病毒(African swine fever virus,ASFV)能导致猪群高死亡率,从而造成严重的经济损失。因此,建立严密、高效的防控系统,包括灵敏、准确的诊断方法以及高效的预报预警机制等,以避免ASF传入我国。本文旨在建立一种新型而高灵敏度的ASFV微滴数字PCR(Droplet digital PCR,dd PCR)检测方法。【方法】针对ASFV K205R基因设计一对特异性引物和Taq Man探针,通过反应条件优化,建立了ASFV实时荧光定量PCR(q PCR)和dd PCR检测方法;并对两种方法的线性关系、灵敏性、特异性和重复性进行了评估,利用建立的ASFV检测方法对163份国内和进境的组织样本及血清样本进行检测,血清样本经商品化ASFV ELISA试剂盒复检,评估不同方法检测结果的符合率。【结果】建立的ASFV q PCR和dd PCR检测方法线性关系较好(R2≥0.998),dd PCR的最低检测限度可达到0.36拷贝,在20μL反应体系中约为10拷贝/反应,检测灵敏度是q PCR的10倍。ASFV dd PCR检测方法具有很高的特异性和重复性,不与其他猪常见病毒发生交叉反应。【结论】该方法灵敏度高、特异性强,可作为一种有效的分子生物学方法来诊断ASFV,为防止该病传入我国以及ASFV的实时监测提供新的技术储备,同时促进dd PCR技术在我国的发展和应用。     

Novel primers for detection and quantification of Myxococcus species in situ

A nested polymerase chain reaction (PCR) protocol using unique primers was developed to detect and quantify Myxococcus species from environmental samples. The protocol amplified most species of Myxococcus when 10 pg of DNA representing 1000 cells was present, although over half were amplified with as little as 0.1 pg (10 cells). The protocol did not amplify other myxobacterial species, members of the δ‐proteobacteria or other unrelated organisms tested at significantly higher concentrations of DNA. The primers were also used in quantitative PCRs, which accurately estimated the population levels in soil.     

A PCR-based method for detection and quantification of small RNAs

Recent cloning efforts have identified hundreds of thousands of small RNAs including micro RNAs (miRNAs), Piwi-interacting RNAs (piRNAs), and small nucleolar RNAs (snoRNAs). These non-coding small RNAs need to be further validated and characterized by detecting and quantifying their expression in different tissues and during different developmental courses. A simple, accurate, and sensitive method for small RNA expression profiling is in high demand. Here, we report such a PCR-based method.     

PCR-based procedures for detection and quantification of Staphylococcus aureus and their application in food

AIMS: To evaluate the specificity of nuc targeted primers for PCR detection of Staphylococcus aureus in different food matrices and to establish a RTQ-PCR procedure suitable for the routine detection and quantification of this pathogen in food. METHODS AND RESULTS: Specificity of nuc targeted primers (Pri1-Pri2 and the newly designed RTQ-PCR primers) was tested on a total of 157 strains of genetically confirmed identity, including reference and food isolates. PCR detection on artificially inoculated beef samples by DNA extraction using a DNeasy Tissue Kit (Qiagen GmhH, Hilden, Germany) showed a sensitivity value around 10(3) CFU g(-1). The two RTQ-PCR systems, incorporating SYBR-Green I and TaqMan, respectively, applied in the present work improved the sensitivity of conventional PCR by lowering the detection level to 10 and 100 cells, respectively. Out of 164 naturally contaminated foods tested for the presence of Staph. aureus, 74 were positive by conventional PCR and 69 by the traditional culture method with a high degree of result agreement between both methodologies (93.3%). CONCLUSIONS: PCR approaches, using nuc targeted primers, have proved specific and combined with growth techniques may improve detection of Staph. aureus in different types of food. SIGNIFICANCE AND IMPACT OF THE STUDY: The SYBR-Green I real-time PCR approach established allows the sensitive, automated and quantitative detection of Staph. aureus for routine analysis at a reasonable cost.     

A rapid and quantitative method for the detection of Leptospira species in human leptospirosis

Prompt laboratory diagnosis of leptospirosis infection facilitates patient management and initiation of therapy. A cost effective real-time PCR assay using SYBR Green I was developed for detection of pathogenic leptospires in serum specimens. Specific PCR products were obtained only with DNA of pathogenic Leptospira genomospecies. LightCycler PCR ability to distinguish between species was possible using melting curves, providing an approach for identification with a specific Tm assigned to a single species or set of species. Assay sensitivity was approximately 50 leptospires/ml, corresponding to one to two genome copies in a PCR mixture. Fifty-one patients who had clinical symptoms consistent with leptospirosis were tested both with a previously described rrs amplification and our real-time assay. Our LFB1 real-time assay confirmed the diagnosis for 25 patients (49%, 25/51) and revealed an estimated density of 8.0x10(1)-3.9x10(4) leptospires/ml of blood. The total assay time for 12 clinical samples from sample to data analysis was less than 3 h. These data illustrate the potential of our LFB1 real-time assay for the rapid detection of leptospires in serum samples and their subsequent quantification in a single run.     

A TaqMan-based real-time PCR assay for the specific detection and quantification of Leuconostoc mesenteroides in meat products

A new real-time PCR procedure was developed for the specific detection and quantification of Leuconostoc mesenteroides in meat products. It is a TaqMan assay based on 23S rRNA gene targeted primers and probe. Specificity was evaluated using purified DNA from 132 strains: 102 lactic acid bacteria (LAB), including 57 reference strains and 46 food isolates, belonging to genus Leuconostoc and related genera, and 30 non-LAB strains. Quantification was linear over at least 5 log units using both serial dilutions of purified DNA and calibrated cell suspensions from Leuconostoc mesenteroides ssp. dextranicum CECT 912T. This assay was able to detect at least five genomic equivalents, using purified DNA or 59 CFU per reaction when using calibrated cell suspensions. It performed successfully when tested on an artificially inoculated meat product, with a minimum threshold of 10(4) CFU g(-1) for the accurate quantification of Leuconostoc mesenteroides.     

A real-time polymerase chain reaction assay for quantification of allele ratios and correction of amplification bias

Allele-specific epigenetic modifications are crucial for several important biological functions, including genomic imprinting and X-inactivation in mammals. Consequently, an ever increasing number of investigations requires accurate quantification of the relative abundance of parental alleles of a specific sequence in a DNA sample. Here, combining the use of polymorphic restriction sites with real-time polymerase chain reaction (PCR) amplification, we describe a simple and quantitative assay to measure allele ratios. The efficiency of the assay was assessed on genomic DNA for several polymorphic restriction sites located in the mouse Igf2/H19 imprinted locus. The assay was also successfully applied to quantify allele ratio in cDNA samples. In addition, we provide an experimental procedure for detection and correction of potential PCR amplification bias which significantly extends the range of application of the assay.     

Primer and probe sets for group-specific quantification of the genera Nitrosomonas and Nitrosospira using real-time PCR

Use of quantitative real-time PCR (QPCR) with TaqMan probes is increasingly popular in various environmental works to detect and quantify a specific microorganism or a group of target microorganism. Although many aspects of conducting a QPCR assay have become very easy to perform, a proper design of oligonucleotide sequences comprising primers and a probe is still considered as one of the most important aspects of a QPCR application. This work was conducted to design group specific primer and probe sets for the detection of ammonia oxidizing bacteria (AOB) using a real-time PCR with a TaqMan system. The genera Nitrosomonas and Nitrosospira were grouped into five clusters based on similarity of their 16S rRNA gene sequences. Five group-specific AOB primer and probe sets were designed. These sets separately detect four subgroups of Nitrosomonas (Nitrosomonas europaea-, Nitrosococcus mobilis-, Nitrosomonas nitrosa-, and Nitrosomonas cryotolerans-clusters) along with the genus Nitrosospira. Target-group specificity of each primer and probe set was initially investigated by analyzing potential false results in silico, followed by a series of experimental tests for QPCR efficiency and detection limit. In general, each primer and probe set was very specific to the target group and sensitive to detect target DNA as low as two 16S rRNA gene copies per reaction mixture. QPCR efficiency, higher than 93.5%, could be achieved for all primer and probe sets. The primer and probe sets designed in this study can be used to detect and quantify the beta-proteobacterial AOB in biological nitrification processes and various environments.     

Evaluation of a shortened QIAsymphony DNA extraction protocol for stool samples using a multiplex real-time PCR for the detection of enteric pathogens

A shortened DNA extraction protocol for the QIAsymphony SP was evaluated by quantitative and qualitative comparison of real-time PCR results of 150 co-extracted stool samples. The average ?Cycle threshold value for positive pathogenic targets was 0.28 Ct. A consensus of 96.91%, with a correlation coefficient of 0.9880 was recorded.     

Specific detection and quantification of the marine flavobacterial genus Zobellia on macroalgae using novel qPCR and CARD-FISH assays

The flavobacterial genus Zobellia is considered as a model to study macroalgal polysaccharide degradation. The lack of data regarding its prevalence and abundance in coastal habitats constitutes a bottleneck to assess its ecological strategies. To overcome this issue, real-time quantitative PCR (qPCR) and fluorescence in situ hybridization (FISH) methods targeting the 16S rRNA gene were optimized to specifically detect and quantify Zobellia on the surface of diverse macroalgae. The newly designed qPCR primers and FISH probes targeted 98 and 100% of the Zobellia strains in silico and their specificity was confirmed using pure bacterial cultures. The dynamic range of the qPCR assay spanned 8 orders of magnitude from 10 to 10⁸ 16S rRNA gene copies and the detection limit was 0.01% relative abundance of Zobellia in environmental samples. Zobellia-16S rRNA gene copies were detected on all surveyed brown, green and red macroalgae, in proportion varying between 0.1 and 0.9% of the total bacterial copies. The absolute and relative abundance of Zobellia varied with tissue aging on the kelp Laminaria digitata. Zobellia cells were successfully visualized in Ulva lactuca and stranded Palmaria palmata surface biofilm using CARD-FISH, representing in the latter 10⁵ Zobellia cells·cm⁻² and 0.43% of total bacterial cells. Overall, qPCR and CARD-FISH assays enabled robust detection, quantification and localization of Zobellia representatives in complex samples, underlining their ecological relevance as primary biomass degraders potentially cross-feeding other microorganisms.     

Survival of a novel endophytic fungus Phomopsis liquidambari B3 in the indole-contaminated soil detected by real-time PCR and its effects on the indigenous microbial community

The recently isolated fungal strain Phomopsis liquidambari B3 can degrade high concentrations of indole, indicating its potential for the bioremediation of indole-contaminated soil. In this study, a specific real-time PCR was developed to detect the survival of P. liquidambari B3 in soil. Subsequently, degradation activity of strain B3 and its effects on indigenous microbial community were analyzed. Results showed the amount of P. liquidambari B3 genomic DNA increased to a maximum 5.67 log (pg g⁻¹ dry soil) 10 days after inoculation of 5.04 log (pg g⁻¹ dry soil), and then gradually decreased with time and after 40 days it was below the detection limit. By the end of the experiment (day 40), bioaugmented microsoms showed a 93.7% decrease in indole, while the values for biostimulated and control microcosms were much lower. Higher microbial biomass and enzyme activities were observed in bioaugmented soil. Denaturing gradient gel electrophoresis analysis showed bioaugmentation increased richness of resident microbial community. These results indicate that P. liquidambari B3 is effective for the remediation of indole-contaminated soil and also provides valuable information about the behavior of the inoculant population during bioremediation, which could be directly used in the risk assessment of inoculant population and optimization of bioremediation process.     

Species-specific DNA probe and development of a quantitative PCR assay for the detection of Morganella morganii

Aims: To develop a SYBR Green quantitative PCR assay (qPCR) for the specific detection of Morganella morganii, a fish pathogen responsible for the Histamine Fish Poisoning. Methods and Results: A new primer set, amplifying a 179‐bp fragment of the 16S rRNA gene, was selected for specificity, and 14 M. morganii strains and 32 non‐Morganella strains were evaluated. The melting temperature of 84°C was consistently specific for the amplicon. Two standard curves were constructed: the minimum detection sensitivity was 0·563 pg of pure DNA, corresponding to DNA extracted from nine cells of M. morganii. The qPCR assay was evaluated in experiments with seeded fish samples, and the regression coefficient values were calculated. Conclusions: A highly specific and rapid assay was developed for the detection of M. morganii in tuna fish samples. Significance and Impact of the Study: This method represents the first study about the quantification of pathogenic M. morganii in fish products. This approach can be utilized to prevent the presence of this undesirable species in the food chain.     

Optimizing techniques to capture and extract environmental DNA for detection and quantification of fish

Few studies have examined capture and extraction methods for environmental DNA (eDNA) to identify techniques optimal for detection and quantification. In this study, precipitation, centrifugation and filtration eDNA capture methods and six commercially available DNA extraction kits were evaluated for their ability to detect and quantify common carp (Cyprinus carpio) mitochondrial DNA using quantitative PCR in a series of laboratory experiments. Filtration methods yielded the most carp eDNA, and a glass fibre (GF) filter performed better than a similar pore size polycarbonate (PC) filter. Smaller pore sized filters had higher regression slopes of biomass to eDNA, indicating that they were potentially more sensitive to changes in biomass. Comparison of DNA extraction kits showed that the MP Biomedicals FastDNA SPIN Kit yielded the most carp eDNA and was the most sensitive for detection purposes, despite minor inhibition. The MoBio PowerSoil DNA Isolation Kit had the lowest coefficient of variation in extraction efficiency between lake and well water and had no detectable inhibition, making it most suitable for comparisons across aquatic environments. Of the methods tested, we recommend using a 1.5 μm GF filter, followed by extraction with the MP Biomedicals FastDNA SPIN Kit for detection. For quantification of eDNA, filtration through a 0.2–0.6 μm pore size PC filter, followed by extraction with MoBio PowerSoil DNA Isolation Kit was optimal. These results are broadly applicable for laboratory studies on carps and potentially other cyprinids. The recommendations can also be used to inform choice of methodology for field studies.