Short Communication: Ageratum,Croton and Malvastrum harbour geminiviruses: evidence through PCR amplification

Ageratum conyzoides, Croton bonpladianum and Malvastrum coromandelianum are common weeds found around agricultural fields. In several cases these were found to exhibit vein yellowing and yellow mosaic symptoms. Using degenerate primers specific for whitefly-transmitted geminiviruses (WTGs), and total DNA isolated from such infected plants (exhibiting the above symptoms) as a template, 1.2kbp fragments were amplified and were shown to have homology to DNA-A of Indian tomato leaf curl virus (ITLCV) by Southern hybridization. In control experiments the same primers failed to amplify any DNA fragments from the total DNA isolated from healthy plants (no symptoms as above). These results show that Ageratum, Croton and Malvastrum harbour geminivirus(es).     

Polycation-assisted DNA detection by reduction triggered fluorescence amplification probe

We have developed a fluorescence detection system for DNA, assisted by a comb-type cationic polymer (PLL-g-DX), for accelerating the reaction turnover. The combination of fluorogenic DNA probes with a comb-type cationic polymer has been demonstrated to be an effective means of signal amplification during the detection process. The method described herein represents a simple and enzyme-free detection.     

Multi-gene amplification: simultaneous detection of three virulence genes in diarrhoeal stool

Enterotoxigenic Escherichia coli (ETEC) and Shigella account for a substantial proportion of acute diarrhoeal illnesses among Third-World children. Rapid detection of these infectious agents in faeces followed by the prompt implementation of public health measures could help reduce their spread during the early phase of epidemics. Towards this end, three pairs of synthetic oligonucleotide primers were prepared and shown to hybridize specifically to the genes encoding the heat-stable (ST) and the heat-labile (LT) enterotoxins of ETEC and to invasion-associated loci (ial) of the large Shigella virulence plasmid. When the three primer pairs were used together in the polymerase chain reaction (PCR), the three corresponding genetic loci could be simultaneously amplified using DNA extracted directly from stool; the amplified products were readily detected by ST-, LT- and ial-specific, alkaline phosphatase-labelled oligonucleotide probes (AP probes). The performance of this system was evaluated in a Mayan community in southeastern Mexico, where diarrhoeal illnesses are a common cause of childhood morbidity and mortality. Using only simple and inexpensive laboratory equipment, multigene amplification with these primers and probes led to the identification of ETEC and/or Shigella in the stools of 20 out of 71 children with diarrhoea; the procedure could be completed in seven hours and was more sensitive than conventional diagnostic tests or DNA probes used without amplification.     

Label-free optical bifunctional oligonucleotide probe for homogeneous amplification detection of disease markers

Oligonucleotide-based detection schemes that avoid chemical modification possess significant advantages, including simplified design, intrinsic affinity for targets, low cost and ease to extend applications. In this contribution, we developed a label-free self-locked bifunctional oligonucleotide probe (signaling probe) for the detection of different disease markers in parallel. Two signal enhancement techniques based on isothermal circular strand-displacement polymerization reaction, cyclical nucleic acid strand-displacement polymerization (CNDP) and cyclical common (nonnucleic acid) target-displacement polymerization (CCDP), were employed to implement the amplification assay for p53 gene and PDGF-BB, respectively. The attractive assay properties confirmed the effectiveness of isothermal polymerization in common biosensing systems without evolving any chemical modification: PDGF could be detected down to 0.87ng/mL, and a dynamic response range of 8-5000ng/mL was achieved; The capability to screen the p53 gene was also considerably improved, including the detection limit, sensitivity, dynamic range and so on. Moreover, because no any chemical modification of the signaling probe was acquired and different targets were separately detected in homogeneous solution. This interrogating platform exhibits the design flexibility, convenience, simplicity and cost-effectiveness. The success achieved here is expected to serve as a significant step toward the development of robust label-free oligonucleotide probes in biomarker profiling and disease diagnostics.     

PCR amplification and simultaneous digoxigenin incorporation of long DNA probes for fluorescence in situ hybridization.

Nonradioactive in situ hybridization has found widespread applications in cytogenetics. Basic requirements are DNA probes in sufficient amounts and of high specificity as well as a labeling protocol of good reproducibility. The PCR has been of fundamental importance for the amplification of DNA sequences and thus for the production of DNA probes. Meanwhile, PCR protocols for amplification of DNA have reached a high degree of automation. So far, incorporation of labeled nucleotides into these DNA probes has normally been done by nick translation. Here we show that in using the PCR, amplification of a DNA probe larger than one kilobase accompanied by simultaneous incorporation of digoxigenin-11-dUTP can be performed for in situ hybridization experiments. As an example, the DNA probe pUC 1.77 specific for the subcentromeric region q12 of chromosome number 1 was used and hybridized against metaphase chromosomes from human lymphocytes. The labeled chromosome region was detected by anti-digoxigenin-fluorescein, Fab fragments. The experiments were evaluated by digital image analysis of microphotographs.     

Multiplex real-time PCR assay for simultaneous detection of Omphalotus guepiniformis and Lentinula edodes

A rapid multiplex real-time PCR assay was developed to achieve highly specific, simultaneous detection of two kinds of mushrooms, Omphalotus guepiniformis and Lentinula edodes. Primers and TaqMan minor groove binder probes were designed according to the internal transcribed spacers 1-5.8S region of rDNA and evaluated by the specificity for fruiting bodies of 17 O. guepiniformis, 16 L. edodes and samples from 57 other species. DNA extracts of all the target species had positive signals with no cross-reaction, the limit of detection being 0.00025 ng of DNA. Threshold cycle (Ct) values for raw and processed fruiting bodies and for fruiting bodies (1% (w/w)) mixed with foodstuffs or artificial gastric juice contents ranged from 17.16 to 26.60 for both examined species. This new assay proved specific to the target species, highly sensitive, and applicable to processed food samples and gastric juice contents, making it useful for rapidly identifying O. guepiniformis and L. edodes.     

MegaPlex PCR: a strategy for multiplex amplification

'MegaPlex PCR' is a robust technology for highly multiplexed amplification of specific DNA sequences. It uses target-specific pairs of PCR primers that are physically separated by surface immobilization. Initial surface-based amplification cycles are then coupled to efficient solution-phase PCR using one common primer pair. We demonstrate this method by co-amplifying and genotyping 75 unselected human single-nucleotide polymorphism (SNP) loci.     

Exponential ATP amplification through simultaneous regeneration from AMP and pyrophosphate for luminescence detection of bacteria

Bacteria monitoring is essential for many industrial manufacturing processes, particularly those involving in food, biopharmaceuticals, and semiconductor production. Firefly luciferase ATP luminescence assay is a rapid and simple bacteria detection method. However, the detection limit of this assay for Escherichia coli is approximately 10⁴ colony-forming units (CFU), which is insufficient for many applications. This study aims to improve the assay sensitivity by simultaneous conversion of PP_i and AMP, two products of the luciferase reaction, back to ATP to form two chain-reaction loops. Because each consumed ATP continuously produces two new ATP molecules, this approach can achieve exponential amplification of ATP. Two consecutive enzyme reactions were employed to regenerate AMP into ATP: adenylate kinase converting AMP into ADP using UTP as the energy source, and acetate kinase catalyzing acetyl phosphate and ADP into ATP. The PP_i-recycling loop was completed using ATP sulfurylase and adenosine 5′ phosphosulfate. The modification maintains good quantification linearity in the ATP luminescence assay and greatly increases its bacteria detection sensitivity. This improved method can detect bacteria concentrations of fewer than 10 CFU. This exponential ATP amplification assay will benefit bacteria monitoring in public health and manufacturing processes that require high-quality water.     

Development and evaluation of a multiplex PCR for simultaneous detection of five foodborne pathogens

Aims: To develop a rapid multiplex PCR method for simultaneous detection of five major foodborne pathogens (Staphylococcus aureus, Listeria monocytogenes, Escherichia coli O157:H7, Salmonella Enteritidis and Shigella flexneri, respectively). Methods and Results: Amplification by PCR was optimized to obtain high efficiency. Sensitivity and specificity assays were investigated by testing different strains. With a multipathogen enrichment, multiplex PCR assay was able to simultaneously detect all of the five organisms in artificially contaminated pork samples. The developed method was further applied to retail meat samples, of which 80% were found to be positive for one or more of these five organisms. All the samples were confirmed by traditional culture methods for each individual species. Conclusions: This study reported a rapid multiplex PCR assay using five primers sets for detection of multiple pathogens. Higher consistency was obtained between the results of multiplex PCR and traditional culture methods. Significance and Impact of the Study: This work has developed a reliable, useful and cost‐effective multiplex PCR method. The assay performed equally as well as the traditional cultural method and facilitated the sensitive detection both in artificially contaminated and naturally contaminated samples.     

DNA probe functionalized QCM biosensor based on gold nanoparticle amplification for Bacillus anthracis detection

The rapid detection of Bacillus anthracis, the causative agent of anthrax disease, has gained much attention since the anthrax spore bioterrorism attacks in the United States in 2001. In this work, a DNA probe functionalized quartz crystal microbalance (QCM) biosensor was developed to detect B. anthracis based on the recognition of its specific DNA sequences, i.e., the 168 bp fragment of the Ba813 gene in chromosomes and the 340 bp fragment of the pag gene in plasmid pXO1. A thiol DNA probe was immobilized onto the QCM gold surface through self-assembly via Au-S bond formation to hybridize with the target ss-DNA sequence obtained by asymmetric PCR. Hybridization between the target DNA and the DNA probe resulted in an increase in mass and a decrease in the resonance frequency of the QCM biosensor. Moreover, to amplify the signal, a thiol-DNA fragment complementary to the other end of the target DNA was functionalized with gold nanoparticles. The results indicate that the DNA probe functionalized QCM biosensor could specifically recognize the target DNA fragment of B. anthracis from that of its closest species, such as Bacillus thuringiensis, and that the limit of detection (LOD) reached 3.5 × 10(2)CFU/ml of B. anthracis vegetative cells just after asymmetric PCR amplification, but without culture enrichment. The DNA probe functionalized QCM biosensor demonstrated stable, pollution-free, real-time sensing, and could find application in the rapid detection of B. anthracis.     

Development of a multiplex PCR for the simultaneous amplification and genotyping of glycoprotein N among human cytomegalovirus strains

Genomic variation among human cytomegalovirus (HCMV) strains is probably involved in HCMV-induced pathogenesis. The envelope glycoprotein N (gN) showed extensive genetic polymorphism as HCMV isolates have been clustered into four distinct gN variants (gN-1, gN-2, gN-3, gN-4) whose distribution has been analyzed worldwide using different methodological approaches (PCR-RFLP, PCR-Cloning, PCR-Sequencing). This paper describes a new method for concurrent detection of gN genotypes among HCMV strains using a multiplex gN-variants specific PCR plus visualization on agarose gel, avoiding subsequent steps such as cloning, restriction or sequencing. This novel approach will reduce costs and shorten the detection time of gN polymorphisms among HCMV clinical isolates.     

扩增内标及其在食源性致病菌PCR检测中的应用

虽然PCR技术不断地得到了发展和改进,但检测结果容易出现假阴性而影响检测准确性的现象一直没有得到很好的解决。现在大多数学者普遍认为,在PCR体系中加入扩增内标(即一段人工构建合成的DNA序列或者是一段致病菌的看家基因序列)能有效指示假阴性现象的出现,是PCR检测技术标准化的措施之一。本文将从PCR检测方法中假阴性出现的原因、扩增内标的构建以及扩增内标在PCR检测方面的应用三方面进行综合评述,并结合本实验室的工作基础,介绍扩增内标的简捷构建过程和应用要点,希望在不影响检测灵敏度的前提下,发挥扩增内标对假阴性的指示作用。     

Multiplex PCR for simultaneous detection of five virulence hemolysin genes in Vibrio anguillarum

A multiplex PCR was developed for detection of hemolysin-producing Vibrio anguillarum using primers targeting five hemolysin genes (vah1, vah2, vah3, vah4 and vah5). This method was successful in amplifying reactions containing as little as 100 fg of genomic template DNA. The direct detection of V. anguillarum in clinical specimens by this multiplex PCR was also successful in reactions containing as few as 10 bacterial cells. This multiplex PCR method can be a rapid and sensitive method for detecting pathogenic V. anguillarum.     

PCR microfluidic devices for DNA amplification

The miniaturization of biological and chemical analytical devices by micro-electro-mechanical-systems (MEMS) technology has posed a vital influence on such fields as medical diagnostics, microbial detection and other bio-analysis. Among many miniaturized analytical devices, the polymerase chain reaction (PCR) microchip/microdevices are studied extensively, and thus great progress has been made on aspects of on-chip micromachining (fabrication, bonding and sealing), choice of substrate materials, surface chemistry and architecture of reaction vessel, handling of necessary sample fluid, controlling of three or two-step temperature thermocycling, detection of amplified nucleic acid products, integration with other analytical functional units such as sample preparation, capillary electrophoresis (CE), DNA microarray hybridization, etc. However, little has been done on the review of above-mentioned facets of the PCR microchips/microdevices including the two formats of flow-through and stationary chamber in spite of several earlier reviews [Zorbas, H. Miniature continuous-flow polymerase chain reaction: a breakthrough? Angew Chem Int Ed 1999; 38 (8):1055–1058; Krishnan, M., Namasivayam, V., Lin, R., Pal, R., Burns, M.A. Microfabricated reaction and separation systems. Curr Opin Biotechnol 2001; 12:92–98; Schneegaβ, I., Köhler, J.M. Flow-through polymerase chain reactions in chip themocyclers. Rev Mol Biotechnol 2001; 82:101–121; deMello, A.J. DNA amplification: does ‘small’ really mean ‘efficient’? Lab Chip 2001; 1: 24N–29N; Mariella, Jr. R. MEMS for bio-assays. Biomed Microdevices 2002; 4 (2):77–87; deMello AJ. Microfluidics: DNA amplification moves on. Nature 2003; 422:28–29; Kricka, L.J., Wilding, P. Microchip PCR. Anal BioAnal Chem 2003; 377:820–825]. In this review, we survey the advances of the above aspects among the PCR microfluidic devices in detail. Finally, we also illuminate the potential and practical applications of PCR microfluidics to some fields such as microbial detection and disease diagnosis, based on the DNA/RNA templates used in PCR microfluidics. It is noted, especially, that this review is to help a novice in the field of on-chip PCR amplification to more easily find the original papers, because this review covers almost all of the papers related to on-chip PCR microfluidics.     

Application of broad-range 16S rRNA PCR amplification and DGGE fingerprinting for detection of tick-infecting bacteria

Ticks play an important role in the transmission of arthropod-borne diseases of viral, protozoal and bacterial origin. The present article describes a molecular-biological based method, which facilitated the broad-range analyses of bacterial communities in ixodid ticks (Ixodes ricinus). DNA was extracted both from single ticks and pooled adult ticks. Eubacterial 16S rRNA gene fragments (16S rDNA) were amplified by polymerase chain reaction (PCR) with broad-range ribosomal primers. Sequences spanning the hypervariable V3 region of the 16S rDNA and representing individual bacterial taxons were separated by denaturing gradient gel electrophoresis (DGGE). For phylogenetic identification, DGGE bands were exised, cloned and sequenced. In addition, we set up a 16S rDNA clone library which was screened by DGGE. Sequences were compared with sequences of known bacteria listed in the GenBank database. A number of bacteria were affiliated with the genera Rickettsia, Bartonella, and Borrelia, which are known to be pathogenic and transmitted by ticks. Two sequences were related to the yet to be cultivated Haemobartonella. To our knowledge, Haemobartonella has never been directly detected in I. ricinus. In addition, members of the genera Staphylococcus, Rhodococcus, Pseudomonas, and Moraxella were detected, which have not been identified in ticks so far. Two bacteria were most closely related to a rickettsial endosymbiont of an Acanthamoeba sp., and to an endosymbiont (Legionellaceae, Coxiella group) of the microarthropod Folsomia candida. The results prove that 16S rDNA genotyping in combination with DGGE analysis is a promising approach for the detection and identification of bacteria infecting ticks, regardless of whether these bacteria are fastidious, obligate intracellular or noncultivable.     

Development of real-time PCR array for simultaneous detection of eight human blood-borne viral pathogens

Background

Real-time PCR array for rapid detection of multiple viral pathogens should be highly useful in cases where the sample volume and the time of testing are limited, i.e. in the eligibility testing of tissue and organ donors.

Findings

We developed a real-time PCR array capable of simultaneously detecting eight human viral pathogens: human immunodeficiency virus types 1 and 2 (HIV-1 and -2), hepatitis B virus (HBV), hepatitis C virus (HCV), human T-cell leukemia virus-1 and -2 (HTLV-1 and -2), vaccinia virus (VACV) and West Nile virus (WNV). One hundred twenty (120) primers were designed using a combination of bioinformatics approaches, and, after experimental testing, 24 primer sets targeting eight viral pathogens were selected to set up the array with SYBR Green chemistry. The specificity and sensitivity of the virus-specific primer sets selected for the array were evaluated using analytical panels with known amounts of viruses spiked into human plasma. The array detected: 10 genome equivalents (geq)/ml of HIV-2 and HCV, 50 geq of HIV-1 (subtype B), HBV (genotype A) and WNV. It detected 100–1,000 geq/ml of plasma of HIV-1 subtypes (A – G), group N and CRF (AE and AG) isolates. Further evaluation with a panel consisting of 28 HIV-1 and HIV-2 clinical isolates revealed no cross-reactivity of HIV-1 or HIV-2 specific primers with another type of HIV. All 28 viral isolates were identified with specific primer sets targeting the most conserved genome areas. The PCR array correctly identified viral infections in a panel of 17 previously quantified clinical plasma samples positive for HIV-1, HCV or HBV at as low as several geq per PCR reaction.

Conclusions

The viral array described here demonstrated adequate performance in the testing of donors’ clinical samples. Further improvement in its sensitivity for the broad spectrum of HIV-1 subtypes is under development.     

Molecular Zipper: a fluorescent probe for real-time isothermal DNA amplification

Rolling-circle amplification (RCA) and ramification amplification (RAM, also known as hyperbranched RCA) are isothermal nucleic acid amplification technologies that have gained a great application in in situ signal amplification, DNA and protein microarray assays, single nucleotide polymorphism detection, as well as clinical diagnosis. Real-time detection of RCA or RAM products has been a challenge because of most real-time detection systems, including Taqman and Molecular Beacon, are designed for thermal cycling-based DNA amplification technology. In the present study, we describe a novel fluorescent probe construct, termed molecular zipper, which is specially designed for quantifying target DNA by real-time monitoring RAM reactions. Our results showed that the molecular zipper has very low background fluorescence due to the strong interaction between two strands. Once it is incorporated into the RAM products its double strand region is opened by displacement, therefore, its fluorophore releases a fluorescent signal. Applying the molecular zipper in RAM assay, we were able to detect as few as 10 molecules within 90 min reaction. A linear relationship was observed between initial input of targets and threshold time (R² = 0.985). These results indicate that molecular zipper can be applied to real-time monitoring and qualification of RAM reaction, implying an amenable method for automatic RAM-based diagnostic assays.