枣疯病植原体实时荧光定量PCR检测方法的研究

目的:建立枣疯病植原体拷贝数检测实时荧光定量PCR方法,为枣疯病植原体定量检测提供技术支持。方法:构建质粒标准品,设计实时荧光PCR探针引物,优化体系,建立标准曲线,并进行重复性验证。结果:制备了枣疯病植原体标准质粒,建立了稳定的质粒标准品检测体系(R2=0.998,检测限10拷贝,定量限100拷贝)。结论:实时荧光定量PCR检测方法重复性好,可用于枣疯病植原体的拷贝数检测,为枣疯病植原体检验检测和病害防治提供了技术支持。     

Suitability of Quantitative Real-Time PCR To Estimate the Biomass of Fungal Root Endophytes

A nested single-copy locus-based quantitative PCR (qPCR) assay and a multicopy locus-based qPCR assay were developed to estimate endophytic biomass of fungal root symbionts belonging to the Phialocephala fortinii sensu lato-Acephala applanata species complex (PAC). Both assays were suitable for estimation of endophytic biomass, but the nested assay was more sensitive and specific for PAC. For mycelia grown in liquid cultures, the correlation between dry weight and DNA amount was strong and statistically significant for all three examined strains, allowing accurate prediction of fungal biomass by qPCR. For mycelia colonizing cellophane or Norway spruce roots, correlation between biomass estimated by qPCR and microscopy was strain dependent and was affected by the abundance of microsclerotia. Fungal biomass estimated by qPCR and microscopy correlated well for one strain with poor microsclerotia formation but not for two strains with high microsclerotia formation. The accuracy of qPCR measurement is constrained by the variability of cell volumes, while the accuracy of microscopy can be hampered by overlapping fungal structures and lack of specificity for PAC. Nevertheless, qPCR is preferable because it is highly specific for PAC and less time-consuming than quantification by microscopy. There is currently no better method than qPCR-based quantification using calibration curves obtained from pure mycelia to predict PAC biomass in substrates. In this study, the DNA amount of A. applanata extracted from 15 mm of Norway spruce fine root segments (mean diameter, 610 μm) varied between 0.3 and 45.5 ng, which corresponds to a PAC biomass of 5.1 ± 4.5 μg (estimate ± 95% prediction interval) and 418 ± 264 μg.Interactions between fungi and plants are very common in nature and range from mutualistic to pathogenic (41). The outcome of a plant-fungus interaction largely depends on the extent of colonization by the fungus, independent of whether pathogenic or mutualistic fungal species are involved (6, 8). This is also true for endophytic species. In contrast to infections by pathogenic fungi, where disease symptoms are expressed after a comparatively short period of incubation, infection by endophytic fungi does not cause disease symptoms for prolonged periods, because once inside the tissue, endophytes assume a quiescent state either for the whole lifetime of the infected plant tissue or until the host is adversely affected by the arrival of biotic or abiotic stress (34, 38, 42, 45). Therefore, switching endophyte behavior from neutral to pathogenic or mutualistic can depend on the predisposition of the host tissue, environmental factors, and the extent of colonization. For instance, in conifer needles, the biomass of endophytic Rhabdocline parkeri thalli increase over time (44). It has been postulated that the needles die as soon as the endophyte''s biomass exceeds a certain threshold value (42). Therefore, attainment of the threshold usually coincides with natural senescence. However, the threshold can either be lowered or reached prematurely if host resistance is reduced by adverse factors. Thus, the health status of plants depends on the density of colonization by the endophyte, and vice versa.Estimation of the extent of colonization is difficult, and there are certain conditions that must be met for techniques to determine fungal biomass. First, they should reproducibly combine target (i.e., species/genotype) specificity with accurate quantification of biomass. Traditionally, microscopy has been used to measure hyphal length or proportional colonization of host tissue (4, 28, 30). However, determination of fungal biomass by microscopy is very laborious, and results vary between investigators. Moreover, visual quantification is unspecific, as species designation is often difficult or impossible. Chemical methods measuring the amount of specific biomolecules stored inside fungal cells or released into the environment (e.g., the fatty acid ergosterol or the carbohydrate chitin) are also widely used (13, 49). Although these methods are much less laborious than microscopy, they are nonspecific and problems can arise if used for field samples (33), and the minimum sample size required is comparatively high (14, 31). Real-time quantitative PCR (qPCR) (23, 43) combines specificity at different taxonomic levels with accurate measurement of DNA copy number and allows quantification of DNA in very small samples. Different qPCR chemistries (TaqMan, SYBR green, or molecular beacons) and methods are available (22, 32). The choice of the locus used for qPCR assays largely depends on the aim of the study. While multicopy genes allow the detection of lower DNA amounts, single-copy genes give more precise measurements of DNA copy number, as the number of repeats of multicopy loci can differ between strains and even within a single individual strain (7, 24). In addition, sensitivity of qPCR can be increased by applying a nested approach, where the entire locus is initially amplified by conventional PCR, and the resulting product is then quantified with the specific primer-probe combination in a second step (11, 39). Diagnostic qPCR assays have been used for early and rapid detection of plant pathogens in the environment and in diseased tissues (9, 32, 50). However, little investigation has been done into the usefulness of qPCR to estimate fungal biomass, and considerable disagreement exists. For example, qPCR biomass estimates of multinucleate arbuscular mycorrhizal fungi (AMF) correlated only poorly with estimates obtained by visual assessment (12). This was proposed to be due to the multinucleate nature of Glomeromycota. On the other hand, a correlation between qPCR and mycelial dry weight was demonstrated for the Swiss needle cast-causing parasite Phaeocryptopus gaeumannii (52) and between qPCR and hyphal length for the ectomycorrhizal fungus (EMF) Piloderma croceum (36). Partial correspondence between ergosterol assays and qPCR has been demonstrated in needles infected with P. gaeumannii (51), and a high correlation between qPCR and ergosterol was found in tissues colonized by the conifer root pathogen Heterobasidion annosum (25).In the present study we tested the suitability of qPCR to estimate the biomass of a common group of ascomycetous root endophytes. Members of the Phialocephala fortinii sensu lato-Acephala applanata species complex (PAC) (17, 46) are ubiquitous root symbionts in woody plants, especially in conifers and heathland shrubs, where they are the most prominent endophytes (1). PAC form loose networks of hyphae running mostly parallel to the root axis on the root surface but also grow inter- and intracellularly within the root cortex (47). Inside cortical cells and under certain culture conditions, PAC species can form microsclerotia, which are tight complexes of more or less isodiametric, to irregular, thick-walled cells that can endure harsh conditions and may therefore serve as resting spores and units of dispersal (2, 37, 53). The ecological role of PAC members is still controversial, despite several studies (21). The effects of PAC on their hosts were described as being pathogenic in some studies but beneficial in others. This variability in behavior was mainly due to the use of different, undefined isolates and a multitude of experimental designs which either favored PAC members or the host plants (21). Recently, isolates characterized by specific molecular markers have become available, making PAC-host interaction studies more meaningful.In this study we aimed (i) to develop a specific qPCR method that allows detection of all PAC members, (ii) to test the method''s suitability for biomass estimation in three different experimental systems in vitro (liquid fungal cultures, cellophane culture, and colonized roots of Picea abies seedlings) by using biomass estimates obtained by microscopy as reference, and (iii) to compare the reproducibility, sensitivity, and specificity of a nested single-copy qPCR assay and a multicopy qPCR assay.     

实时荧光定量PCR的数据分析方法

实时荧光定量PCR是目前检测目的核酸拷贝数及分析靶基因在mRNA表达水平相对变化的主流技术。研究表明,分析结果的准确性依赖于数据分析方法的可靠性。我们简要综述实时荧光定量PCR的数据分析方法。     

实时荧光定量PCR技术及其应用

定量PCR的问世是继定性PCR(即常规PCR)后分子生物学方法学研究的一大飞跃,实现了对核酸信息量的分析比较,为疾病的诊断和治疗提供了更多、更有效的基因水平信息。本文介绍实时荧光定量PCR技术及其在兽医领域中的应用,包括近年来研究和应用较多的几种定量PCR技术。     

实时荧光定量PCR技术的研究进展与应用

实时荧光定量PCR技术(real-time fluorescent quantitative PCR,FQ-PCR)以其特异性强、灵敏度高、重复性好、定量准确、自动化程度高、速度快、全封闭反应等优点在人类和动物疾病的快速检测、食品安全检测、定量分析、基因分型、基因表达研究、以及疫苗效力测定中成为分子生物学研究的重要工具...     

实时荧光定量PCR的发展和数据分析

实时荧光定量PCR技术是基因时代一项用于检测mRNA的常用技术,是临床检测和基础研究中不可缺少的重要研究方法,包括绝对定量PCR和相对定量PCR。该技术的特点是可以减少PCR后操作,在比较不同浓度的mRNA方面具有非常宽的动力学范围。我们就目前实时荧光定量PCR的发展及数据的分析进行综述。     

Internally Controlled Real-Time PCR Method for Quantitative Species-Specific Detection and vapA Genotyping of Rhodococcus equi

We developed a novel quantitative real-time PCR (Q-PCR) method for the soil actinomycete Rhodococcus equi, an important horse pathogen and emerging human pathogen. Species-specific quantification was achieved by targeting the chromosomal monocopy gene choE, universally conserved in R. equi. The choE Q-PCR included an internal amplification control (IAC) for identification of false negatives. A second Q-PCR targeted the virulence plasmid gene vapA, carried by most horse isolates but infrequently found in isolates from other sources. The choE-IAC and vapA assays were 100% sensitive and specific as determined using 178 R. equi isolates, 77 nontarget bacteria, and a panel of 60 R. equi isolates with known vapA⁺ and vapA-negative (including vapB⁺) plasmid genotypes. The vapA⁺ frequency among isolate types was as follows: horse, 85%; human, 20%; bovine and pig, 0%; others, 27%. The choE-IAC Q-PCR could detect up to one genome equivalent using R. equi DNA or 100 bacteria/ml using DNA extracted from artificially contaminated horse bronchoalveolar lavage (BAL) fluid. Quantification was linear over a 6-log dynamic range down to ≈10 target molecules (or 1,000 CFU/ml BAL fluid) with PCR efficiency E of >0.94. The vapA assay had similar performance but appeared unsuitable for accurate (vapA⁺) R. equi quantification due to variability in target gene or plasmid copy number (1 to 9). The dual-reaction Q-PCR system here reported offers a useful tool to both medical and veterinary diagnostic laboratories for the quantitative detection of R. equi and (optional) vapA⁺ “horse-pathogenic” genotype determination.     

Quantitative Real-Time PCR Assays To Identify and Quantify Fecal Bifidobacterium Species in Infants Receiving a Prebiotic Infant Formula

A healthy intestinal microbiota is considered to be important for priming of the infants' mucosal and systemic immunity. Breast-fed infants typically have an intestinal microbiota dominated by different Bifidobacterium species. It has been described that allergic infants have different levels of specific Bifidobacterium species than healthy infants. For the accurate quantification of Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium dentium, Bifidobacterium infantis, and Bifidobacterium longum in fecal samples, duplex 5′ nuclease assays were developed. The assays, targeting rRNA gene intergenic spacer regions, were validated and compared with conventional PCR and fluorescent in situ hybridization methods. The 5′ nuclease assays were subsequently used to determine the relative amounts of different Bifidobacterium species in fecal samples from infants receiving a standard formula or a standard formula supplemented with galacto- and fructo-oligosaccharides (OSF). A breast-fed group was studied in parallel as a reference. The results showed a significant increase in the total amount of fecal bifidobacteria (54.8% ± 9.8% to 73.4% ± 4.0%) in infants receiving the prebiotic formula (OSF), with a diversity of Bifidobacterium species similar to breast-fed infants. The intestinal microbiota of infants who received a standard formula seems to resemble a more adult-like distribution of bifidobacteria and contains relatively more B. catenulatum and B. adolescentis (2.71% ± 1.92% and 8.11% ± 4.12%, respectively, versus 0.15% ± 0.11% and 1.38% ± 0.98% for the OSF group). In conclusion, the specific prebiotic infant formula used induces a fecal microbiota that closely resembles the microbiota of breast-fed infants also at the level of the different Bifidobacterium species.     

Quantitative Detection of Listeria monocytogenes in Biofilms by Real-Time PCR

A quantitative method based on a real-time PCR assay to enumerate Listeria monocytogenes in biofilms was developed. The specificity for L. monocytogenes of primers targeting the listeriolysin gene was demonstrated using a SYBR Green I real-time PCR assay. The number of L. monocytogenes detected growing in biofilms was 6 × 10² CFU/cm².     

Quantitative Detection of Clostridium tyrobutyricum in Milk by Real-Time PCR

We developed a real-time PCR assay for the quantitative detection of Clostridium tyrobutyricum, which has been identified as the major causal agent of late blowing in cheese. The assay was 100% specific, with an analytical sensitivity of 1 genome equivalent in 40% of the reactions. The quantification was linear (R² > 0.9995) over a 5-log dynamic range, down to 10 genome equivalents, with a PCR efficiency of >0.946. With optimized detergent treatment and enzymatic pretreatment of the sample before centrifugation and nucleic acid extraction, the assay counted down to 300 C. tyrobutyricum spores, with a relative accuracy of 82.98 to 107.68, and detected as few as 25 spores in 25 ml of artificially contaminated raw or ultrahigh-temperature-treated whole milk.     

Quantitative Detection of Corynebacterium casei in Cheese by Real-Time PCR

The flora on the surface of smear-ripened cheeses is composed of numerous species of bacteria and yeasts that contribute to the production of the desired organoleptic properties. Due to the absence of selective media, it is very difficult to quantify cheese surface bacteria, and, consequently, the ecology of the cheese surface microflora has not been extensively investigated. We developed a SYBR green I real-time PCR method to quantify Corynebacterium casei, a major species of smear-ripened cheeses, using primers designed to target the 16S rRNA gene. It was possible to recover C. casei genomic DNA from the cheese matrix with nearly the same yield that C. casei genomic DNA is recovered from cells recovered by centrifugation from liquid cultures. Quantification was linear over a range from 10⁵ to 10¹⁰ CFU per g of cheese. The specificity of the assay was demonstrated with DNA from species related to C. casei and from other bacteria and yeasts belonging to the cheese flora. Nine commercial cheeses were analyzed by real-time PCR, and six of them were found to contain more than 10⁵ CFU equivalents of C. casei per g. In two of them, the proportion of C. casei in the total bacterial flora was nearly 40%. The presence of C. casei in these samples was further confirmed by single-strand conformation polymorphism analysis and by a combined approach consisting of plate counting and 16S rRNA gene sequencing. We concluded that SYBR green I real-time PCR may be used as a reliable species-specific method for quantification of bacteria from the surface of cheeses.     

Quantitative Real-Time PCR using the Thermo Scientific Solaris qPCR Assay

The Solaris qPCR Gene Expression Assay is a novel type of primer/probe set, designed to simplify the qPCR process while maintaining the sensitivity and accuracy of the assay. These primer/probe sets are pre-designed to >98% of the human and mouse genomes and feature significant improvements from previously available technologies. These improvements were made possible by virtue of a novel design algorithm, developed by Thermo Scientific bioinformatics experts.Several convenient features have been incorporated into the Solaris qPCR Assay to streamline the process of performing quantitative real-time PCR. First, the protocol is similar to commonly employed alternatives, so the methods used during qPCR are likely to be familiar. Second, the master mix is blue, which makes setting the qPCR reactions easier to track. Third, the thermal cycling conditions are the same for all assays (genes), making it possible to run many samples at a time and reducing the potential for error. Finally, the probe and primer sequence information are provided, simplifying the publication process.Here, we demonstrate how to obtain the appropriate Solaris reagents using the GENEius product search feature found on the ordering web site (www.thermo.com/solaris) and how to use the Solaris reagents for performing qPCR using the standard curve method.Download video file.^{(108M, mp4)}     

Real-Time Quantitative PCR Measurement of Ileal Lactobacillus salivarius Populations from Broiler Chickens To Determine the Influence of Farming Practices

A real-time quantitative PCR assay targeting a 16S-23S intergenic spacer region sequence was devised to measure the sizes of populations of Lactobacillus salivarius present in ileal digesta collected from broiler chickens. This species has been associated with deconjugation of bile salts in the small bowel and reduced broiler productivity. The assay was tested as a means of monitoring the sizes of L. salivarius populations from broilers fed diets with different compositions, maintained at different stocking densities, or given the antimicrobial drugs bacitracin and monensin in the feed. Stocking densities did not influence the numbers of L. salivarius cells in the ileum. A diet containing meat and bone meal reduced the size of the L. salivarius population relative to that of chickens given the control diet, as did administration of bacitracin and monensin in the feed. These changes in the target bacterial population were associated with improved broiler weight gain.     

Rapid Detection of Ceratocystis platani Inoculum by Quantitative Real-Time PCR Assay

Ceratocystis platani is the causal agent of canker stain of plane trees, a lethal disease able to kill mature trees in one or two successive growing seasons. The pathogen is a quarantine organism and has a negative impact on anthropogenic and natural populations of plane trees. Contaminated sawdust produced during pruning and sanitation fellings can contribute to disease spread. The goal of this study was to design a rapid, real-time quantitative PCR assay to detect a C. platani airborne inoculum. Airborne inoculum traps (AITs) were placed in an urban setting in the city of Florence, Italy, where the disease was present. Primers and TaqMan minor groove binder (MGB) probes were designed to target cerato-platanin (CP) and internal transcribed spacer 2 (ITS2) genes. The detection limits of the assay were 0.05 pg/μl and 2 fg/μl of fungal DNA for CP and ITS, respectively. Pathogen detection directly from AITs demonstrated specificity and high sensitivity for C. platani, detecting DNA concentrations as low as 1.2 × 10⁻² to 1.4 × 10⁻² pg/μl, corresponding to ∼10 conidia per ml. Airborne inoculum traps were able to detect the C. platani inoculum within 200 m of the closest symptomatic infected plane tree. The combination of airborne trapping and real-time quantitative PCR assay provides a rapid and sensitive method for the specific detection of a C. platani inoculum. This technique may be used to identify the period of highest risk of pathogen spread in a site, thus helping disease management.     

Quantitative Real-Time Legionella PCR for Environmental Water Samples: Data Interpretation

Quantitative Legionella PCRs targeting the 16S rRNA gene (specific for the genus Legionella) and the mip gene (specific for the species Legionella pneumophila) were applied to a total of 223 hot water system samples (131 in one laboratory and 92 in another laboratory) and 37 cooling tower samples (all in the same laboratory). The PCR results were compared with those of conventional culture. 16S rRNA gene PCR results were nonquantifiable for 2.8% of cooling tower samples and up to 39.1% of hot water system samples, and this was highly predictive of Legionella CFU counts below 250/liter. PCR cutoff values for identifying hot water system samples containing >10³ CFU/liter legionellae were determined separately in each laboratory. The cutoffs differed widely between the laboratories and had sensitivities from 87.7 to 92.9% and specificities from 77.3 to 96.5%. The best specificity was obtained with mip PCR. PCR cutoffs could not be determined for cooling tower samples, as the results were highly variable and often high for culture-negative samples. Thus, quantitative Legionella PCR appears to be applicable to samples from hot water systems, but the positivity cutoff has to be determined in each laboratory.     

Evaluation of a Rapid, Quantitative Real-Time PCR Method for Enumeration of Pathogenic Candida Cells in Water

Quantitative PCR (QPCR) technology, incorporating fluorigenic 5′ nuclease (TaqMan) chemistry, was utilized for the specific detection and quantification of six pathogenic species of Candida (C. albicans, C. tropicalis, C. krusei, C. parapsilosis, C. glabrata and C. lusitaniae) in water. Known numbers of target cells were added to distilled and tap water samples, filtered, and disrupted directly on the membranes for recovery of DNA for QPCR analysis. The assay's sensitivities were between one and three cells per filter. The accuracy of the cell estimates was between 50 and 200% of their true value (95% confidence level). In similar tests with surface water samples, the presence of PCR inhibitory compounds necessitated further purification and/or dilution of the DNA extracts, with resultant reductions in sensitivity but generally not in quantitative accuracy. Analyses of a series of freshwater samples collected from a recreational beach showed positive correlations between the QPCR results and colony counts of the corresponding target species. Positive correlations were also seen between the cell quantities of the target Candida species detected in these analyses and colony counts of Enterococcus organisms. With a combined sample processing and analysis time of less than 4 h, this method shows great promise as a tool for rapidly assessing potential exposures to waterborne pathogenic Candida species from drinking and recreational waters and may have applications in the detection of fecal pollution.     

实时荧光定量PCR及其在传染性疾病检测中的应用

实时荧光定量PCR技术被广泛应用于实验研究、临床检测中。与普通的PCR相比,实时荧光定量PCR技术具有特异性强、灵敏度高、重复性好、定量准确、速度快、全封闭反应等优点。我们综述了实时荧光定量PCR技术的原理、定量方法,及其在传染性疾病检测研究中的应用。     

Detection of Legionellae in Hospital Water Samples by Quantitative Real-Time LightCycler PCR

Contamination of hospital water systems with legionellae is a well-known cause of nosocomial legionellosis. We describe a new real-time LightCycler PCR assay for quantitative determination of legionellae in potable water samples. Primers that amplify both a 386-bp fragment of the 16S rRNA gene from Legionella spp. and a specifically cloned fragment of the phage lambda, added to each sample as an internal inhibitor control, were used. The amplified products were detected by use of a dual-color hybridization probe assay design and quantified with external standards composed of Legionella pneumophila genomic DNA. The PCR assay had a sensitivity of 1 fg of Legionella DNA (i.e., less than one Legionella organism) per assay and detected 44 Legionella species and serogroups. Seventy-seven water samples from three hospitals were investigated by PCR and culture. The rates of detection of legionellae were 98.7% (76 of 77) by the PCR assay and 70.1% (54 of 77) by culture; PCR inhibitors were detected in one sample. The amounts of legionellae calculated from the PCR results were associated with the CFU detected by culture (r = 0.57; P < 0.001), but PCR results were mostly higher than the culture results. Since L. pneumophila is the main cause of legionellosis, we further developed a quantitative L. pneumophila-specific PCR assay targeting the macrophage infectivity potentiator (mip) gene, which codes for an immunophilin of the FK506 binding protein family. All but one of the 16S rRNA gene PCR-positive water samples were also positive in the mip gene PCR, and the results of the two PCR assays were correlated. In conclusion, the newly developed Legionella genus-specific and L. pneumophila species-specific PCR assays proved to be valuable tools for investigation of Legionella contamination in potable water systems.