A comparison of nucleic acid amplification techniques for the assessment of bacterial viability

AIMS: The ability to determine the presence and viability status of bacteria by molecular methods could offer significant advantages to the food, environmental and health sectors, in terms of improved speed and sensitivity of detection. METHODS AND RESULTS: In this study, we have assessed three amplification techniques, PCR, RT-PCR and NASBA, for their ability to detect nucleic acid persistence in an E. coli strain following heat-killing. NASBA offered the greatest sensitivity of the three methods tested. The presence of residual DNA and mRNA could be detected by PCR and NASBA, respectively, for up to 30 h postdeath, by which time cell death had been confirmed by culture methods. Thus a single quantitative measurement based on nucleic acid amplification did not permit unequivocal determination of cell viability. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The correlation between cell viability and persistence of nucleic acids must be well characterized for a particular analytical situation before molecular techniques can be substituted for traditional culture methods.     

Methods for the isolation and identification of Listeria spp. and Listeria monocytogenes: a review

Listeria monocytogenes is an important food-borne pathogen and is widely tested for in food, environmental and clinical samples. Identification traditionally involved culture methods based on selective enrichment and plating followed by the characterization of Listeria spp. based on colony morphology, sugar fermentation and haemolytic properties. These methods are the gold standard; but they are lengthy and may not be suitable for testing of foods with short shelf lives. As a result more rapid tests were developed based on antibodies (ELISA) or molecular techniques (PCR or DNA hybridization). While these tests possess equal sensitivity, they are rapid and allow testing to be completed within 48 h. More recently, molecular methods were developed that target RNA rather than DNA, such as RT-PCR, real time PCR or nucleic acid based sequence amplification (NASBA). These tests not only provide a measure of cell viability but they can also be used for quantitative analysis. In addition, a variety of tests are available for sub-species characterization, which are particularly useful in epidemiological investigations. Early typing methods differentiated isolates based on phenotypic markers, such as multilocus enzyme electrophoresis, phage typing and serotyping. These phenotypic typing methods are being replaced by molecular tests, which reflect genetic relationships between isolates and are more accurate. These new methods are currently mainly used in research but their considerable potential for routine testing in the future cannot be overlooked.     

Self-sustained sequence replication (3SR): an alternative to PCR

 The amplification of target nucleic acids before hybridization is one of the most powerful approaches for the detection of low copy number RNA and DNA. The best known amplification reaction is PCR which has many applications. However, certain drawbacks of the PCR reaction provide a role for alternative amplification methods. One of these methods is the self-sustained sequence replication (3SR) reaction, which is an isothermal method for RNA amplification depending on the action of three enzymes. 3SR has been used in several in vitro applications and has also been modified for in situ use (IS-3SR). We have studied IS-3SR with the measles virus as a model and have found that it can significantly amplify the amount of intracellular RNA. Such a level of amplification could raise the amount of single copy RNA to the level of detection by conventional in situ hybridization. Although careful controls to insure its specificity must be carried out, IS-3SR has several advantages, including ease of use, preserved cell morphology, and specificity for RNA amplification, which make it an attractive alternative to the in situ PCR method. Accepted: 27 June 1997     

Extracting nucleic acids from activated sludge which reflect community population diversity

Critical to most studies in molecular microbial ecology is the application of DNA/RNA extraction methods which can reveal the true level of population biodiversity present in samples from the community under investigation. Activated sludge communities have been studied extensively using molecular methods, but rarely have the nucleic acid isolation methods applied been assessed for their ability to achieve this. This study compares eight published RNA and DNA extraction protocols and one commercially available DNA isolation kit for their capacity to provide high quality nucleic acids that reflect the community composition. Each method was assessed on the basis of nucleic acid yield, purity and integrity, and the ability to provide PCR amplifiable RNA and DNA from known marker populations that varied in their resistance to nucleic acid extraction. Only three consistently provided DNA from each of the marker populations known to be present in the samples from fluorescence in situ hybridisation analysis. The failure of the other methods emphasises the need to validate all DNA/RNA extraction protocols. It is recommended that several validated extraction methods be used and the extracts pooled to further minimise any risk of bias.     

Redefining relativity: quantitative PCR at low template concentrations for industrial and environmental microbiology

The application of PCR techniques in environmental and industrial microbiology is complicated by innumerable organic and inorganic contaminants and enzyme inhibitors that copurify with nucleic acids. These complications are compounded in quantitative PCR (qPCR) methods, which are predicated upon subtle yet significant assumptions of amplification efficiency and the representativeness of the sample with respect to the environment or industrial process from which it was obtained. In low-biomass and/or low-template situations, additional concerns related to target gene spatial heterogeneity in the sample, differential DNA (or RNA) extraction efficiency, molecular sampling error, attenuation of PCR inhibitors and amplification bias can quickly undermine fundamental assumptions of conventional competitive PCR (cPCR) and most-probable-number PCR (MPN-PCR) formats. A critical evaluation of cPCR and MPN-PCR assumptions is therefore presented within the context of environmental microbiology and low-template enumerations. Fundamental conclusions from the analysis of qPCR assumptions are that: (a) environmental qPCR enumerations are invariably estimates, not absolute enumerations, which are relative to the PCR standard; (b) traditional cPCR assays are ill-suited for environmental applications, especially in low-biomass situations; and (c) both cPCR and traditional MPN-PCR practices insufficiently account for field-scale, process-level or experimental variations that arise and become amplified in PCR enumerations. Thus, sample representativeness and errors related to sample replication are frequently more important than errors related to the qPCR assay itself. Based upon this critique of qPCR assumptions, an alternate qPCR method for routine environmental application is described which is based upon replicative limiting dilution analysis and the pragmatic tradeoffs between analytical sensitivity and practical utility. Received 9 February 1998/ Accepted in revised form 26 June 1998     

Pre-PCR processing

Polymerase chain reaction (PCR) is recognized as a rapid, sensitive, and specific molecular diagnostic tool for the analysis of nucleic acids. However, the sensitivity and kinetics of diagnostic PCR may be dramatically reduced when applied directly to biological samples, such as blood and feces, owing to PCR-inhibitory components. As a result, pre-PCR processing procedures have been developed to remove or reduce the effects of PCR inhibitors. Pre-PCR processing comprises all steps prior to the detection of PCR products, that is, sampling, sample preparation, and deoxyribonucleic acid (DNA) amplification. The aim of pre-PCR processing is to convert a complex biological sample with its target nucleic acids/cells into PCR-amplifiable samples by combining sample preparation and amplification conditions. Several different pre-PCR processing strategies are used: (1) optimization of the DNA amplification conditions by the use of alternative DNA polymerases and/or amplification facilitators, (2) optimization of the sample preparation method, (3) optimization of the sampling method, and (4) combinations of the different strategies. This review describes different pre-PCR processing strategies to circumvent PCR inhibition to allow accurate and precise DNA amplification.     

Development of conventional and real-time NASBA for the detection of Legionella species in respiratory specimens

Isothermal nucleic acid sequence-based amplification (NASBA) was applied to detect Legionella 16S rRNA. The assay was originally developed as a Legionella pneumophila conventional NASBA assay with electrochemiluminescence (ECL) detection and was subsequently adapted to a L. pneumophila real-time NASBA format and a Legionella spp. real-time NASBA using molecular beacons. L. pneumophila RNA prepared from a plasmid construct was used to assess the analytical sensitivity of the assay. The sensitivity of the NASBA assay was 10 molecules of in vitro wild type L. pneumophila RNA and 0.1-1 colony-forming units (CFU) of L. pneumophila. In spiked respiratory specimens, the sensitivity of the NASBA assays was 1-10000 CFU of L. pneumophila serotype 1 depending on the background. After dilution of the nucleic acid extract prior to amplification, 1-10 CFU of L. pneumophila serotype 1 could be detected with both detection methods. Finally, 27 respiratory specimens, well characterized by culture and PCR, collected during a L. pneumophila outbreak, were tested by conventional and real-time NASBAs. All 11 PCR positive samples were positive by conventional NASBA, 9/11 and 10/11 were positive by L. pneumophila real-time NASBA and Legionella spp. real-time NASBA, respectively.     

Development of a molecular diagnosis assay based on electrohybridization at plastic electrodes and subsequent PCR

Technologies enabling specific recognition of medically relevant nucleic acid sequences will play a pivotal role in future medical diagnosis. Whereas many approaches to molecular diagnosis systems include DNA microarrays on chips and fluorometric detection, the basis of our approach is the use of inexpensive components like plastic or metal thin film electrodes with low multiplexing and an electrochemical detection unit. To increase the sensitivity, PCR can be used as an intermediate step. For selective enrichment, specific nucleic acid probes were covalently attached at their 5′-ends to conducting polycarbonate/carbon fiber electrodes. Complementary oligonucleotides were enriched at the electrodes by cyclic inversion of an electrochemical potential, transferred into a PCR vial and thermally or electrochemically desorbed. The analysis of the PCR product shows the efficiency and selectivity of the electrochemical enrichment. Hybridization of DNA was shown by electrochemical methods, in this work especially by differential pulse voltammetry (DPV) using the single strand specific hybridization redox indicator osmium(VIII)-tetroxide, and potentiometric stripping analysis (PSA). This combination of experimental methods is the basis for a molecular diagnosis system including a disposable nucleic acid modified working electrode for specific enrichment, detection and quantification, and an optional capillary PCR module for fast amplification.     

快速PCR研究进展

PCR是最常用的分子生物学技术之一,通过变性、退火和延伸的循环来完成核酸分子的大量扩增。快速PCR就是基于普通PCR的工作原理,在保证PCR反应特异性、灵敏性、保真度的前提下,在更短时间内完成对核酸分子的扩增。近年来已经开展了许多有关方面的研究工作。本文将以DNA聚合酶的改进、添加剂的选择、热循环仪改进为重点内容,综述快速PCR技术的研究进展。     

PCR detection of Cryptosporidium parvum in environmental samples—a review of published protocols and current developments

Since 1991 more than 30 PCR protocols have been published, which show a potential to replace the current microscopic detection method for Cryptosporidium parvum in environmental samples and food. This review provides a synoptic comparison of these protocols with respect to the following features: isolation and purification of oocysts from tested matrices, elimination of free DNA, viability and infectivity assessment, release of nucleic acids, nucleic acid extraction, type of PCR (PCR, RT-PCR, internal-standard-PCR, in situ PCR, TaqMan-PCR), primary product detection, additional specificity control, secondary product detection, reported sensitivity, cross-reaction with other Cryptosporidium species, and target and sequence information such as amplicon length, primer sequences, multiple copy target, presence of strain-specific differences in the amplicon, GenBank accession numbers and gene function. The results demonstrate that problems like PCR inhibition, viability assessment, and the requirement of an extreme sensitivity have been solved. PCR assays would be most valuable to control presence-absence standards in defined matrix volumes, and the setup of such standards would very much contribute to a rapid introduction of this awaited technology into routine monitoring of environmental, water and food samples, and to a further standardization of the various protocols. It can be expected that satisfactory solutions for quantification will be found for a growing number of PCR-based assays. Systematic field evaluation and interlaboratory studies will complement our present knowledge of these methods in the near future. Received 5 May 1998/ Accepted in revised form 7 September 1998     

DNA from formalin-fixed tissue: extraction or repair? That is the question

Establishing effective DNA-based protocols for use on archival material fixed in formaldehyde (formalin) is a particularly challenging task. Formalin fixation induces cross-linking with nucleic acids and proteins, thereby reducing the amount and quality of the extracted DNA. Previous attempts have primarily focused on optimizing DNA extraction protocols. Here we focus on the use of enzymes capable of in vitro repair of DNA extracts prior to amplification of the nucleic acids by the polymerase chain reaction (PCR). The amplification success of mitochondrial DNA was greater using the repair enzyme assay (56%) than with the regular PCR assay (20%), and even more convincing results were obtained with the amplified nuclear ribosomal region (91% versus 21%). These results indicate that in vitro repair of DNA damage (depurinated sites, strand nicks and base modifications) increases the number of samples that amplify, amplify to a greater extent and amplify fewer ancillary bands and that DNA repair has been overlooked as a way of improving the efficiency of molecular methods used on formalin-fixed samples. Fidelity has not been specifically investigated, but preliminary results indicate that misincorporation is not a major problem.     

Locked nucleic acids in PCR primers increase sensitivity and performance

The incorporation of locked nucleic acids (LNAs) into oligonucleotide primers has been shown to increase template binding strength and specificity for DNA amplification. Real-time PCR and DNA sequencing have been shown to be significantly enhanced by the use of LNAs. Theoretically, increasing primers' binding strength may also increase the sensitivity of conventional PCR, reducing minimum template requirements. We compared LNA-modified PCR primers with their standard DNA counterparts for amplification sensitivity with template amounts as low as 5 pg. Although the results are highly dependent on the design of the LNA primers, large increases in peak height can be achieved from as little as 75 pg, as well as clearer and more complete profiles. Increased amplification success with lower template amounts may also be seen. Additionally, the use of LNAs can enhance multiplexing. Thus, incorporating LNAs into PCR primers can increase amplification success, sensitivity, and performance under a wide range of conditions.     

Alternative Molecular Tests for Virological Diagnosis

Several nucleic acid amplification techniques (NAATs), particularly PCR and real-time PCR, are currently used in the routine clinical laboratories. Such approaches have allowed rapid diagnosis with a high degree of sensitivity and specificity. However, conventional PCR methods have several intrinsic disadvantages such as the requirement for temperature cycling apparatus, and sophisticated and costly analytical equipments. Therefore, amplification at a constant temperature is an attractive alternative method to avoid these requirements. A new generation of isothermal amplification techniques are gaining a wide popularity as diagnostic tools due to their simple operation, rapid reaction and easy detection. The main isothermal methods reviewed here include loop-mediated isothermal amplification, nucleic acid sequence-based amplification, and helicase-dependent amplification. In this review, design criteria, potential of amplification, and application of these alternative molecular tests will be discussed and compared to conventional NAATs.     

Quantitation of viral load using real-time amplification techniques

Real-time PCR amplification techniques are currently used to determine the viral load in clinical samples for an increasing number of targets. Real-time PCR reduces the time necessary to generate results after amplification. In-house developed PCR and nucleic acid sequence-based amplification (NASBA)-based systems combined with several detection strategies are being employed in a clinical diagnostic setting. The importance of these assays in disease management is still in an exploration phase. Although these technologies have the implicit capability of accurately measuring DNA and RNA in clinical samples, issues related to standardization and quality control must be resolved to enable routine implementation of these technologies in molecular diagnostics.     

The use of NASBA for the detection of microbial pathogens in food and environmental samples

The isothermal amplification method nucleic acid sequence-based amplification (NASBA), which amplifies RNA, has been reported as useful for the detection of microbial pathogens in food and environmental samples. Methods have been published for Campylobacter spp., Listeria monocytogenes and Salmonella enterica ser. Enteritidis in various foods and for Cryptosporidium parvum in water. Both 16S rRNA and various mRNAs have been used as target molecules for detection; the latter may have advantages in allowing specific detection of viable cells. Most of the methods to detect pathogens in foods have employed enrichment in nutrient medium prior to NASBA, as this can ensure sensitivity of detection and encourage the detection of only viable target cells. Although a relatively recent method, NASBA has the potential for adoption as a diagnostic tool for environmental pathogens.     

Comparative analysis of eukaryotic marine microbial assemblages from 18S rRNA gene and gene transcript clone libraries by using different methods of extraction

Eukaryotic marine microbes play pivotal roles in biogeochemical nutrient cycling and ecosystem function, but studies that focus on the protistan biogeography and genetic diversity lag-behind studies of other microbes. 18S rRNA PCR amplification and clone library sequencing are commonly used to assess diversity that is culture independent. However, molecular methods are not without potential biases and artifacts. In this study, we compare the community composition of clone libraries generated from the same water sample collected at the San Pedro Ocean Time Series (SPOTs) station in the northwest Pacific Ocean. Community composition was assessed using different cell lysis methods (chemical and mechanical) and the extraction of different nucleic acids (DNA and RNA reverse transcribed to cDNA) to build Sanger ABI clone libraries. We describe specific biases for ecologically important phylogenetic groups resulting from differences in nucleic acid extraction methods that will inform future designs of eukaryotic diversity studies, regardless of the target sequencing platform planned.     

Amplification methods to increase the sensitivity of in situ hybridization: play card(s).

In situ hybridization (ISH) has proved to be an invaluable molecular tool in research and diagnosis to visualize nucleic acids in their cellular environment. However, its applicability can be limited by its restricted detection sensitivity. During the past 10 years, several strategies have been developed to improve the threshold levels of nucleic acid detection in situ by amplification of either target nucleic acid sequences before ISH (e.g., in situ PCR) or the detection signals after the hybridization procedures. Here we outline the principles of tyramide signal amplification using the catalyzed reporter deposition (CARD) technique, present practical suggestions to efficiently enhance the sensitivity of ISH with CARD, and discuss some applications and possible future directions of in situ nucleic acid detection using such an amplification strategy.