Fungal molecular diagnostics: a mini review

Conventional methods to identify fungi have often relied on identification of disease symptoms, isolation and culturing of environmental organisms, and laboratory identification by morphology and biochemical tests. Although these methods are still fundamental there is an increasing move towards molecular diagnostics of fungi in all fields. In this review, some of the molecular approaches to fungal diagnostics based on polymerase chain reaction (PCR) and DNA/RNA probe technology are discussed. This includes several technological advances in PCR-based methods for the detection, identification and quantification of fungi including real-time PCR which has been successfully used to provide rapid, quantitative data on fungal species from environmental samples. PCR and probe based methods have provided new tools for the enumeration of fungal species, but it is still necessary to combine the new technology with more conventional methods to gain a fuller understanding of interactions occurring in the environment. Since its introduction in the mid 1980's PCR has provided many molecular diagnostic tools, some of which are discussed within this review, and with the advances in micro-array technology and real-time PCR methods the future is bright for the development of accurate, quantitative diagnostic tools that can provide information not only on individual fungal species but also on whole communities.     

PCR-based diagnostics for anaerobic infections

Conventional methods to identify anaerobic bacteria have often relied on unique clinical findings, isolation of organisms, and laboratory identification by morphology and biochemical tests (phenotypic tests). Although these methods are still fundamental, there is an increasing move toward molecular diagnostics of anaerobes. In this review, some of the molecular approaches to anaerobic diagnostics based on the polymerase chain reaction (PCR) are discussed. This includes several technological advances in PCR-based methods for the detection, identification, and quantitation of anaerobes including real-time PCR which has been successfully used to provide rapid, quantitative data on anaerobic species on clinical samples. Since its introduction in the mid-1980s, PCR has provided many molecular diagnostic tools, some of which are discussed within this review. With the advances in micro-array technology and real-time PCR methods, the future is bright for the development of accurate, quantitative diagnostic tools that can provide information not only on individual anaerobic species but also on whole communities.     

Molecular investigations of mitochondrial deletions: Evaluating the usefulness of different genetic tests

Deletions in mitochondrial DNA are a common cause of mitochondrial disorders. The molecular diagnosis of mtDNA deletions for years was based on Southern hybridization later replaced by PCR methods such as PCR with primers specific for a particular deletion (mainly the so-called common deletion of 4977bp) and long PCR. In order to evaluate the usefulness of MLPA (Multiplex Ligation-dependent Probe Amplification) in molecular diagnosis of large scale mtDNA deletions we compare four diagnostic methods: Southern hybridization, PCR, long-PCR and MLPA in a group of 16 patients with suspected deletions. Analysis was performed on blood, muscle and in one case hepatic tissue DNA. The MLPA was not able to confirm all the deletions detected by PCR methods, but due to its relative ease of processing, minimal equipment, low costs and the additional possibility to detect frequent point mtDNA mutations in one assay it is worth considering as a screening method. We recommend to always confirm MLPA results by PCR methods.     

Clinical applications of molecular biology for infectious diseases

Molecular biological methods for the detection and characterisation of microorganisms have revolutionised diagnostic microbiology and are now part of routine specimen processing. Polymerase chain reaction (PCR) techniques have led the way into this new era by allowing rapid detection of microorganisms that were previously difficult or impossible to detect by traditional microbiological methods. In addition to detection of fastidious microorganisms, more rapid detection by molecular methods is now possible for pathogens of public health importance. Molecular methods have now progressed beyond identification to detect antimicrobial resistance genes and provide public health information such as strain characterisation by genotyping. Treatment of certain microorganisms has been improved by viral resistance detection and viral load testing for the monitoring of responses to antiviral therapies. With the advent of multiplex PCR, real-time PCR and improvements in efficiency through automation, the costs of molecular methods are decreasing such that the role of molecular methods will further increase. This review will focus on the clinical utility of molecular methods performed in the clinical microbiology laboratory, illustrated with the many examples of how they have changed laboratory diagnosis and therefore the management of infectious diseases.     

食源性致病菌多重分子生物学检测技术研究进展

快速、可靠的食源性致病菌高通量检测方法对于确保食品安全具有重要意义,近年基于DNA水平的多重分子生物学检测技术迅速发展,针对各种不同的食源性致病菌建立了多种多重分子检测技术,包括多重PCR、多重实时荧光PCR以及基因芯片等。对这些多重分子检测技术的最新研究进展作一综述,并且建议在今后该技术的研究中,仍需要在食品中多种致病菌同时选择性增菌培养、亚致死损伤修复以及检测内标的构建等方面取得突破,从而能够更好地实现食源性致病菌的高通量检测。     

分子标记法在昆虫学研究中的应用

比较分析了以Southern杂交技术、PCR及以重复序列为基础的3类分子标记技术。并对各种分子标记的优缺点及其在昆虫学上的应用进行了比较分析,分析探讨了分子生物学在昆虫学中的应用前景。     

Evolution of Toxoplasma-PCR methods and practices: a French national survey and proposal for technical guidelines

The molecular diagnosis of toxoplasmosis lacks standardisation due to the use of numerous methods with variable performance. This diversity of methods also impairs robust performance comparisons between laboratories. The harmonisation of practices by diffusion of technical guidelines is a useful way to improve these performances. The knowledge of methods and practices used for this molecular diagnosis is an essential step to provide guidelines for Toxoplasma-PCR. In the present study, we aimed (i) to describe the methods and practices of Toxoplasma-PCR used by clinical microbiology laboratories in France and (ii) to propose technical guidelines to improve molecular diagnosis of toxoplasmosis. To do so, a yearly self-administered questionnaire-based survey was undertaken in proficient French laboratories from 2008 to 2015, and guidelines were proposed based on the results of those as well as previously published work. This period saw the progressive abandonment of conventional PCR methods, of Toxoplasma-PCR targeting the B1 gene and of the use of two concomitant molecular methods for this diagnosis. The diversity of practices persisted during the study, in spite of the increasing use of commercial kits such as PCR kits, DNA extraction controls and PCR inhibition controls. We also observed a tendency towards the automation of DNA extraction. The evolution of practices did not always go together with an improvement in those, as reported notably by the declining use of Uracil-DNA Glycosylase to avoid carry-over contamination. We here propose technical recommendations which correspond to items explored during the survey, with respect to DNA extraction, Toxoplasma-PCR and good PCR practices.     

A decade with nucleic acid‐based microbiological methods in safety control of foods

In the last decade, nucleic acid‐based methods gradually started to replace or complement the culture‐based methods and immunochemical assays in routine laboratories involved in food control. In particular, real‐time polymerase chain reaction (PCR) was technically developed to the stage of good speed, sensitivity and reproducibility, at minimized risk of carry‐over contamination. Basic advantages provided by nucleic acid‐based methods are higher speed and added information, such as subspecies identification, information on the presence of genes important for virulence or antibiotic resistance. Nucleic acid‐based methods are attractive also to detect important foodborne pathogens for which no classical counterparts are available, namely foodborne pathogenic viruses. This review briefly summarizes currently available or developing molecular technologies that may be candidates for involvement in microbiological molecular methods in the next decade. Potential of nonamplification as well as amplification methods is discussed, including fluorescent in situ hybridization, alternative PCR chemistries, alternative amplification technologies, digital PCR and nanotechnologies.     

Chelex-100法及酚氯仿法提取阴道毛滴虫DNA的比较

目的-比较Chelex-100法和酚氯仿法提取阴道毛滴虫基因组DNA。方法-分别用Chelex-100法和酚氯仿法提取阴道毛滴虫基因组DNA,用PCR法检测DNA提取的有效性。结果两种方法提取的DNA经PCR扩增均有特定的条带。结论-两种方法均能提取阴道毛滴虫DNA。Chelex-100方法简便、省时,较适用于分子生物学研究及临床PCR扩增使用。     

PCR inhibitor levels in concentrates of biosolid samples predicted by a new method based on excitation-emission matrix spectroscopy

Biosolids contain a wide variety of organic contaminants that are known for their ability to inhibit PCR. During sample processing, these contaminants are coconcentrated with microorganisms. Elevated concentrations of these compounds in concentrates render samples unsuitable for molecular applications. Glycine-based elution and recovery methods have been shown to generate samples with fewer PCR inhibitory compounds than the current U.S. EPA-recommended method for pathogen recovery from biosolids. Even with glycine-based methods, PCR inhibitors still persist in concentrations that may interfere with nucleic acid amplification. This results in considerable loss of time and resources and increases the probability of false negatives. A method to estimate the degree of inhibition prior to application of molecular methods is desirable. Here we report fluorescence excitation-emission matrix (EEM) profiling as a tool for predicting levels of molecular inhibition in sample concentrates of biosolids.     

两种新型目标分子标记技术——CDDP与PAAP

本文介绍了CDDP和PAAP这两种新型目标分子标记技术的产生原理、引物设计、PCR扩增、PCR产物检测手段、分子标记特征及带型模式,在此基础上对它们的技术特点和技术优势进行了概括,最后,对它们的应用前景进行了展望。     

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。     

Rapid Diagnosis of Bloodstream Infections with PCR Followed by Mass Spectrometry

Achieving a rapid microbiological diagnosis is crucial for decreasing morbidity and mortality of patients with a bloodstream infection, as it leads to the administration of an appropriate empiric antimicrobial therapy. Molecular methods may offer a rapid alternative to conventional microbiological diagnosis involving blood culture. In this study, the performance of a new technology that uses broad-spectrum PCR coupled with mass spectrometry (PCR/ESI-MS) was evaluated for the detection of microorganisms directly from whole blood. A total of 247 whole blood samples and paired blood cultures were prospectively obtained from 175 patients with a suspicion of sepsis. Both sample types were analyzed using the PCR/ESI-MS technology, and the results were compared with those obtained by conventional identification methods. The overall agreement between conventional methods and PCR/ESI-MS performed in blood culture aliquots was 94.2% with 96.8% sensitivity and 98.5% specificity for the molecular method. When comparing conventional methods with PCR/ESI-MS performed in whole blood specimens, the overall agreement was 77.1% with 50% sensitivity and 93.8% specificity for the molecular method. Interestingly, the PCR/ESI-MS technology led to the additional identification of 13 pathogens that were not found by conventional methods. Using the PCR/ESI-MS technology the microbiological diagnosis of bloodstream infections could be anticipated in about half of the patients in our setting, including a small but significant proportion of patients newly diagnosed. Thus, this promising technology could be very useful for the rapid diagnosis of sepsis in combination with traditional methods.     

Developing molecular diagnostics for detection of red imported fire ants using two genes,Sinv11108 and Sinv11977

Aggressive red imported fire ants (RIFAs) are expanding their habitat due to active international trade and global warming. To prevent infestation and settlement, RIFAs must be removed during the quarantine process. Because RIFAs are social insects and have different morphological characteristics depending on their castes, non‐ant taxonomists have difficulty confirming RIFAs based on their morphological characteristics alone. The disadvantages of previously reported RIFA molecular diagnostics are that they require additional steps, such as restriction enzyme digestion followed by agarose gel electrophoresis separation or DNA sequence verification for polymerase chain reaction (PCR)‐amplified products. To overcome these drawbacks, two RIFA‐specific genes were selected and used to develop diverse PCR‐based RIFA molecular diagnostic techniques. We found that RIFAs could be confirmed by conventional PCR targeting of two RIFA‐specific genes followed by agarose electrophoresis separation. In addition, TaqMan probe real‐time PCR methods had the advantage of confirming RIFAs immediately after the reactions were completed by observing fluorescence indexes. Finally, multiplex PCRs enhanced RIFA specificity and sensitivity. The new molecular diagnostic methods developed in this study had the advantages of reducing false positive and negative results together with high specificity and sensitivity for RIFAs.     

分子生物学方法在食品微生物检测中的应用

近年来,食品安全受到广泛关注。及时、准确地检测出食品中的病原微生物是食品安全检测的重要内容,食品病原微生物的分子生物学检测技术因其特异性和灵敏性而备受瞩目。我们主要介绍了基因探针检测法、PCR检测法和基因芯片检测法的原理、开发及其在食品微生物检测中的应用。     

An advanced molecular strategy to identify bacterial communities on art objects

The application of culture-independent techniques based on molecular biological methods, especially on the PCR amplification of 16S rRNA genes, attempts to overcome some shortcomings of conventional cultivation methods and reveals far more complex bacterial communities on art objects than can be shown by cultivation methods. One of the major challenges of investigating microbial growth on art objects by molecular means is the extraction of DNA, due to small sample amounts and PCR inhibitors. In the present study, we introduce a DNA extraction protocol, which allowed the extraction of PCR-amplifiable DNA from samples derived from lime wall paintings and loamy soil underground. The DNA extracts were used to amplify 16S ribosomal fragments, which were subsequently analyzed by denaturing gradient gel electrophoresis (DGGE). In parallel with the DGGE analysis, clone libraries containing PCR fragments of the ribosomal gene were constructed and clones were screened by DGGE. Clone libraries allow the inclusion of the entire 16S rDNA sequence in the phylogenetic analyses of microorganisms, providing a more reliable phylogenetic identification of microorganisms than is obtained from sequence analyses of excised and directly sequenced DGGE bands.     

微生物生态学中分子生物学方法及T-RFLP技术研究

根据微生物基因 (DNA)多态性来研究微生物的多样性 ,是建立在多聚酶链式反应 (PCR)基础之上分子生物学的新方法 ,克服了传统微生物培养方法的限制。从理论、实验及应用角度出发 ,介绍了几种在微生物生态学中应用较为广泛的分子生物学技术 ;详细阐述了微生物生态学中分子生物学的一种新研究方法---末端限制性片段长度多态性 (T -RFLP)技术 ,该技术作为一种研究微生物群落特征的理想方法已经越来越受到人们的重视。     

Evaluation of two recovery methods for detection of Mycobacterium avium subsp. paratuberculosis by PCR: direct-dilution--centrifugation and C(18)-carboxypropylbetaine processing

A duplex polymerase chain reaction (PCR)-hybridization assay based on Mycobacterium avium subsp. paratuberculosis (MAP)-specific IS900 integration sites was used to evaluate two mycobacterial recovery methods from bovine feces: a direct-dilution-centrifugation method and a C(18)-carboxypropylbetaine (CB-18)-based method. All MAP PCR results were confirmed for absence of inhibitors using a novel PCR system based on the rpoB gene of plant chloroplasts as an internal control. The detection limits of both MAP recovery methods when coupled with PCR were determined to be between 100 and 1000 organisms. Using culture as a 'gold standard' PCR following the direct-dilution-centrifugation protocol was 92.6% sensitive and 83.7% specific, whereas PCR following the CB-18 method was 100% sensitive and 53.5% specific. Both methods were 100% specific when 60 'true' negatives from two uninfected herds were tested. Both the CB-18 and direct processing methods coupled with a target-specific amplification technique may provide greater sensitivity to diagnose subclinical animals as they were able to detect more positives, on samples derived from infected herds, than conventional culture methods; however, more extensive investigation and follow-up of suspect animals will be required to fully validate the MAP recovery and molecular detection protocols described.