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PubMed2000

Pathogen analysis and genetic predisposition testing using microelectronic arrays and isothermal amplification.

BACKGROUND: A simple yet powerful tool for providing for rapid gene identification in the clinic would be the combination of isothermal gene amplification with electronic microchip analysis. This is a first report of such a union of these technologies. METHODS: The first assay demonstrates discrimination between four bacterial pathogens. For this, one portion of the bacterial 16S rRNA gene encompassing a microheterogeneous region was isothermally amplified using Strand Displacement Amplification (SDA). Type identification was then made by "sandwich" assay format either using selective electronic hybridization of amplicons to sequence-specific capture oligonucleotides and a universal, fluorescently labeled reporter oligonucleotide, or, alternatively, sequence-specific reporters and a universal capture oligonucleotide. The second assay tested for the presence or absence of the Factor V Leiden point mutation using DNA obtained from 18 patients in a blind assay. For this, allele-specific SDA was developed. Following amplification using a sense-biotinylated primer and either the corresponding antisense wild type or mutant primer, multiple patient amplicons were targeted to specified locations on the microarray and visualized using a fluorescently labeled reporter oligonucleotide. Positive signals were scored as greater than or equal to two times the background. RESULTS: Bacterial type-specific signals were between 3- to 10-fold greater than nonspecific in both assay formats. Using allele-specific SDA, 100% agreement was observed between PAGE analysis, microarray results, and clinical diagnosis in Factor V mutation analysis. CONCLUSIONS: We demonstrated two model clinical assays combining amplified materials and microelectronic arrays, one potentially suitable for pathogen screening and the other for a deleterious genetic mutation.

Bacteria↗
PubMed2001

One-tube real-time isothermal amplification assay to identify and distinguish human immunodeficiency virus type 1 subtypes A, B, and C and circulating recombinant forms AE and AG.

To halt the human immunodeficiency virus type 1 (HIV-1) epidemic requires interventions that can prevent transmission of numerous HIV-1 subtypes. The most frequently transmitted viruses belong to the subtypes A, B, and C and the circulating recombinant forms (CRFs) AE and AG. A fast one-tube assay that identifies and distinguishes among subtypes A, B, and C and CRFs AE and AG of HIV-1 was developed. The assay amplifies a part of the gag gene sequence of the genome of all currently known HIV-1 subtypes and can identify and distinguish among the targeted subtypes as the reaction proceeds, because of the addition of subtype-specific molecular beacons with multiple fluorophores. The combination of isothermal nucleic acid sequence-based amplification and molecular beacons is a new approach in the design of real-time assays. To obtain a sufficiently specific assay, we developed a new strategy in the design of molecular beacons, purposely introducing mismatches in the molecular beacons. The subtype A and CRF AG isolates reacted with the same molecular beacon. We tested the specificity and sensitivity of the assay on a panel of the culture supernatant of 34 viruses encompassing all HIV-1 subtypes: subtypes A through G, CRF AE and AG, a group O isolate, and a group N isolate. Assay sensitivity on this panel was 92%, with 89% correct subtype identification relative to sequence analysis. A linear relationship was found between the amount of input RNA in the reaction mixture and the time that the reaction became positive. The lower detection level of the assay was approximately 10(3) copies of HIV-1 RNA per reaction. In 38% of 50 serum samples from HIV-1-infected individuals with a detectable amount of virus, we could identify subtype sequences with a specificity of 94% by using sequencing and phylogenetic analysis as the "gold standard." In conclusion, we showed the feasibility of the approach of using multiple molecular beacons labeled with different fluorophores in combination with isothermal amplification to identify and distinguish subtypes A, B, and C and CRFs AE and AG of HIV-1. Because of the low sensitivity, the assay in this format would not be suited for clinical use but can possibly be used for epidemiological monitoring as well as vaccine research studies.

Base Sequence↗
PubMed2006

Isothermal amplification coupled with rapid flow-through hybridisation for sensitive diagnosis of Plum pox virus.

A nucleic acid sequence-based amplification method coupled with rapid flow-through hybridisation (NASBA-FH) was developed for diagnosis of Plum pox virus (PPV). The sensitivity level achieved by NASBA-FH was 10 times higher than that obtained by Co-PCR and 1000 times higher than the sensitivity afforded by RT-PCR. In addition, samples from 262 stone-fruit trees collected during winter and spring seasons were analysed. These samples were tested using methods recommended by the European and Mediterranean Plant Protection Organization to detect PPV (DASI-ELISA, RT-PCR and Co-PCR) and by NASBA-FH. Winter PPV diagnostic results by ELISA and NASBA-FH coincided in 90.8%, while ELISA and PCR-based methods coincided in 91.6% and PCR-based methods with NASBA-FH agreed in 95.4%. In spring, diagnostic results were similar with all the molecular techniques, which agreed with ELISA results for 98.8% of the trees. NASBA-FH was able to detect more positive infections in winter, which were later confirmed in spring. These results indicate that NASBA-FH is a suitable molecular method for routine PPV detection in the winter and spring. This user-friendly isothermal RNA amplification coupled with a very fast flow-through hybridisation (15 min) opens up new possibilities for rapid and reliable diagnosis of a variety of pathogens.

Enzyme-Linked Immunosorbent Assay↗
PubMed2006

Isothermal amplification and molecular typing of the obligate intracellular pathogen Mycobacterium leprae isolated from tissues of unknown origins.

Molecular diagnostic and epidemiology studies require appreciable amounts of high-quality DNA. Molecular epidemiologic methods have not been routinely applied to the obligate intracellular organism Mycobacterium leprae because of the difficulty of obtaining a genomic DNA template from clinical material. Accordingly, we have developed a method based on isothermic multiple-displacement amplification to allow access to a high-quality DNA template. In the study described in this report, we evaluated the usefulness of this method for error-sensitive, multiple-feature molecular analyses. Using test samples isolated from lepromatous tissue, we also evaluated amplification fidelity, genome coverage, and regional amplification bias. The fidelity of amplified genomic material was unaltered; and while regional differences in global amplification efficiency were seen by using comparative microarray analysis, a high degree of concordance of amplified genomic DNA was observed. This method was also applied directly to archived tissue specimens from leprosy patients for the purpose of molecular typing by using short tandem repeats; the success rate was increased from 25% to 92% without the introduction of errors. This is the first study to demonstrate that serial whole-genome amplification can be coupled with error-sensitive molecular typing methods with low-copy-number sequences from tissues containing an obligate intracellular pathogen.

Bacterial Typing Techniques↗
PubMed2003

Isothermal amplification of rabies virus gene.

A sensitive and specific in situ amplification technique is needed to elucidate the dynamics of rabies virus in the body during the long incubation period after infection. To overcome the disadvantage of using the traditional reverse transcription (RT)-PCR in in situ studies, an isothermal nucleic acid sequence-based amplification (NASBA) technique was developed for detection of the rabies virus gene. The NASBA technique involves the use of 4 enzymatic activities to produce multiple RNA copies of the target sequence by means of double-strand cDNA intermediates under an isothermal condition without thermocycling. The amplified cDNA intermediates from the genomic RNA in the rabies virion and the total RNA in the infected cells in NASBA reaction were analyzed by Southern hybridization assays. The specific amplified products of the rabies viral gene with the expected length were detected after 8 hr of incubation in NASBA using both of the RNAs as templates. The NASBA system used in this study was less sensitive than the general RT-PCR technique. This may have been because we employed Southern hybridization for the amplified cDNA intermediates, not many RNA copies, to evaluate the NASBA results. In conclusion, we successfully amplified the rabies viral gene in the NASBA reaction under an isothermal condition. The unique character of this technique would make it particularly valuable for in situ studies not only on rabies virus but also on other RNA viruses.

Animals↗
PubMed2006

Development of loop-mediated isothermal amplification (LAMP)-based SNP markers for shelf-life in melon (Cucumis melo L.).

In this study, LAMP markers linked to shelf-life in melon (Cucumis melo L.) were developed by converting a cleaved amplified polymorphic sequences (CAPS) marker (C2). The CAPS-PCR fragments from the long-shelf-life melon (O-3) and short-shelf-life melon (Nat-2) were cloned and sequenced to construct LAMP primers. A single nucleotide polymorphism (SNP) was identified between O-3 and Nat-2. LAMP primers were designed to detect the SNP. In the LAMP reaction to detect long-shelf-life melon, the turbidity of the templates using O-3, F1, homozygous long-shelf-life F2 lines and heterozygous long-shelf-life F2 lines started to increase after 40 min. In contrast, the turbidity of Nat-2 and homozygous short-shelf-life F2 lines did not increase even after 90 min. In the LAMP reaction to detect short-shelf-life melon, the turbidity of the templates using Nat-2, F1, homozygous short-shelf-life F2 lines and heterozygous long-shelf-life F2 lines started to increase after 40 min. But the turbidity of O-3 and homozygous long-shelf-life F2 lines did not increase after 90 min. This attests to the high reliability and usefulness of LAMP for marker-assisted selection.

Base Sequence↗
PubMed2006

Novel reverse transcription loop-mediated isothermal amplification for rapid detection of foot-and-mouth disease virus.

Speed is paramount in the diagnosis of foot-and-mouth disease (FMD) and simplicity is required if a test is to be deployed in the field. The development of a one-step, reverse transcription loop-mediated amplification (RT-LAMP) assay enables FMD virus (FMDV) to be detected in under an hour in a single tube without thermal cycling. A fragment of the 3D RNA polymerase gene of the virus is amplified at 65 degrees C in the presence of a primer mixture and both reverse transcriptase and Bst DNA polymerase. Compared with real-time RT-PCR, RT-LAMP was consistently faster, and ten copies of FMDV transcript were detected in twenty-two minutes. Amplification products were detected by visual inspection, agarose gel electrophoresis, or in real-time by the addition of a fluorescent dye. The specificity of the reaction was demonstrated by the absence of amplification of RNA from other viruses that cause vesicular diseases and from that of genetically related picornaviruses. Diagnostic sensitivity was validated by the amplification of reference FMDV strains and archival material from field cases of FMD. In comparison with the performance of the established diagnostic TaqMan assay, RT-LAMP appears to be sensitive, rapid, specific, and cost-effective, with the potential for field deployment and use by developing countries for FMDV surveillance.

Animals↗
PubMed2004

An isothermal amplification system based on the tandem-repeated DNA probe.

The hybridization methods and polymerase-based amplification methods are usually employed to detect pathogens and gene expressions quantitatively in clinical practices nowadays. However, the sensitiveness of the former and the specificity of the latter are not yet satisfied. To solve this problem, some promising comprehensive methods have been developed recently. Here we reported a new comprehensive method, a tandem repeated DNA probe-based amplification (TRPBA) system. To establish the TRPBA, TR48, an artificial tandem repeated DNA probe with 48 repeats of a 50 base pair unit was constructed. It could be efficiently amplified by Bst DNA polymerase at 61 degrees C for only 1 h. The products were analyzed either by direct gel electrophoresis or by gel electrophoresis after the digestion with restriction endonuclease HincII. The sensitiveness was as few as 100 copies per test, which was comparable with PCR-based techniques. The TR48 splicing with the DNA fragment of hepatitis B virus used as probe could successfully develop TRPBA to detect hepatitis B virus DNA. The TRPBA can be used in the future to detect many other genes or microorganisms simply by splicing TR48 with their DNA fragments. Thus, TRPBA might be useful because of its sensitiveness and simpleness.

DNA Probes↗
PubMed2005

Evaluation of loop-mediated isothermal amplification (LAMP), PCR and parasitological tests for detection of Trypanosoma evansi in experimentally infected pigs.

Six surra negative piglets (6-week-old) were infected with Trypanosoma evansi and two uninfected piglets were used as negative controls. Detection performances of various diagnostic tests (LAMP, PCR and parasitological tests) were compared by analysing blood samples collected weekly over a period of 11 weeks. With a two by two analysis without a gold standard, all methods were 100% specific. MI had the highest sensitivity of 65%, while LAMP, PCR, MHCT and TBS had sensitivities of 45, 33, 38 and 24%, respectively. However, when the analysis was done using MI as a gold standard, the sensitivity of MHCT was the highest at 53% followed by LAMP, PCR and TBS at 49, 44 and 35%, respectively. All methods gave high specificity above 60%. This study validates LAMP as an alternative method for the diagnosis of surra.

Animals↗
PubMed1997

A molecular predator and its prey: coupled isothermal amplification of nucleic acids.

BACKGROUND: A novel approach to the study of in vitro evolution is provided by the investigation of continuous, functionally coupled, amplifying systems. To date, in vitro evolution experiments have focused on issues of mutation and selection. Our work contributes to the new field of in vitro molecular ecology studies in which detailed information about the relationship between sequence changes and molecular interactions is obtained. Predator-prey systems are interesting in this context both in terms of evolutionary limits and in terms of the potential kinetic properties of oscillation and spatial pattern formation. Such molecular predator-prey models can be extended to a further negative-interaction mode, viral-host molecular evolution. RESULTS: A simple, nonfunctional predator-prey system based on the self-sustained sequence replication reaction is proposed. Coupling within the system is achieved using the single-stranded DNA intermediate of one cycle, the prey cycle, as primer for the second one, the predator cycle. Hybridization by complementary base pairing is the second order reaction step underlying the predation. Single steps of the whole reaction system have been investigated by radiolabeling. Each isolated subsystem operates according to the proposed reaction scheme, and evidence for an efficient coupling of both subsystems according to the proposed mechanism was found. CONCLUSIONS: Simple, interacting model systems based on nucleic acids can be designed and constructed for the study of coevolution. The results of studies such as the one described here will provide a basis for the construction of coupled systems of ribozymes, from which point the engineering of catalytic units for applications in biotechnology is feasible.

Base Sequence↗
PubMed2001

Ramification amplification: a novel isothermal DNA amplification method.

We have developed a novel isothermal DNA amplification method with an amplification mechanism quite different from conventional PCR. This method uses a specially designed circular probe (C-probe) in which the 3' and 5' ends are brought together in juxtaposition by hybridization to a target. The two ends are then covalently linked by a T4 DNA ligase in a target-dependent manner, producing a closed DNA circle. In the presence of an excess of primers (forward and reverse primers), a DNA polymerase extends the bound forward primer along the C-probe and displaces the downstream strand, generating a multimeric single-stranded DNA (ssDNA), analogous to the "rolling circle" replication of bacteriophages in vivo. This multimeric ssDNA then serves as a template for multiple reverse primers to hybridize, extend, and displace downstream DNA, generating a large ramified (branching) DNA complex. This ramification process continues until all ssDNAs become double-stranded, resulting in an exponential amplification that distinguishes itself from the previously described nonexponential rolling circle amplification. In this report, we prove the principle of ramification amplification. By using a unique bacteriophage DNA polymerase, Ø29 DNA Polymerase, that has an intrinsic high processivity, we are able to achieve significant amplification within 1 hour at 35 degrees C. In addition, we applied this technique for in situ detection of Epstein-Barr viral sequences in Raji cells.

Cell Line↗
PubMed1992

Strand displacement amplification--an isothermal, in vitro DNA amplification technique.

Strand Displacement Amplification (SDA) is an isothermal, in vitro nucleic acid amplification technique based upon the ability of HincII to nick the unmodified strand of a hemiphosphorothioate form of its recognition site, and the ability of exonuclease deficient klenow (exo- klenow) to extend the 3'-end at the nick and displace the downstream DNA strand. Exponential amplification results from coupling sense and antisense reactions in which strands displaced from a sense reaction serve as target for an antisense reaction and vice versa. In the original design (G. T. Walker, M. C. Little, J. G. Nadeau and D. D. Shank (1992) Proc. Natl. Acad. Sci 89, 392-396), the target DNA sample is first cleaved with a restriction enzyme(s) in order to generate a double-stranded target fragment with defined 5'- and 3'-ends that can then undergo SDA. Although effective, target generation by restriction enzyme cleavage presents a number of practical limitations. We report a new target generation scheme that eliminates the requirement for restriction enzyme cleavage of the target sample prior to amplification. The method exploits the strand displacement activity of exo- klenow to generate target DNA copies with defined 5'- and 3'-ends. The new target generation process occurs at a single temperature (after initial heat denaturation of the double-stranded DNA). The target copies generated by this process are then amplified directly by SDA. The new protocol improves overall amplification efficiency. Amplification efficiency is also enhanced by improved reaction conditions that reduce nonspecific binding of SDA primers. Greater than 10(7)-fold amplification of a genomic sequence from Mycobacterium tuberculosis is achieved in 2 hours at 37 degrees C even in the presence of as much as 10 micrograms of human DNA per 50 microL reaction. The new target generation scheme can also be applied to techniques separate from SDA as a means of conveniently producing double-stranded fragments with 5'- and 3'-sequences modified as desired.

Base Sequence↗
PubMed2002

[Development of the detection system for Mycobacterium tuberculosis DNA by using the isothermal DNA amplification method ICAN].

The isothermal and chimeric primer-initiated amplification of nucleic acids(ICAN) is a new isothermal DNA amplification method composed of exo- Bca DNA polymerase, RNaseH and DNA-RNA chimeric primers. We developed the detection system, combined the ICAN with luminescence detection by a probe hybridization, for Mycobacterium tuberculosis DNA targeting the IS6110 insertion element. We examined performance tests of the system. This system was able to detect one copy of Mycobacterium tuberculosis DNA for only 3.5 hours, and performance of the system was equivalent or better to the Roche PCR system. We also examined a detection system by using magnetic beads, which system could shorten detection time for 2.5 hours. It was shown that the ICAN system was an efficient and sensitive detection system for Mycobacterium tuberculosis DNA from mass samples.

DNA, Bacterial↗
PubMed2002

Detection of Mycoplasma pneumoniae in spiked clinical samples by nucleic acid sequence-based amplification.

Isothermal nucleic acid sequence-based amplification (NASBA) was applied to the detection of Mycoplasma pneumoniae. M. pneumoniae RNA prepared from a plasmid construct was used to assess the sensitivity of the assay, and an internal control for the detection of inhibitors was constructed. The sensitivity of the NASBA assay was 10 molecules of wild-type M. pneumoniae RNA generated in vitro and 5 color-changing units (CCU) of M. pneumoniae. An appropriate specimen preparation procedure was developed: after protease treatment of the respiratory specimens, guanidine thiocyanate lysis solution (4.7 M guanidine thiocyanate [Sigma-Aldrich NV], 46 mM Tris-HCl [Merck, Darmstadt, Germany], 20 mM EDTA [Sigma-Aldrich NV], 1.2% [wt/vol] Triton X-100 [Sigma-Aldrich NV], pH 6.2.) was added. With spiked throats, nasopharyngeal aspirates, bronchoalveolar lavage specimens, and sputum specimens, the sensitivity of the NASBA assay in the presence of the internal control was 2 x 10(4) molecules of in vitro-generated RNA or 5 CCU of M. pneumoniae. The sensitivity of the NASBA assay was comparable to that of a PCR targeted to the P1 adhesin gene. Fifteen clinical specimens positive for M. pneumoniae by PCR were also positive by NASBA. These results indicate that the sensitivity of detection of M. pneumoniae in spiked respiratory samples by NASBA is high. Together with the use of the internal control, the assay merits evaluation as a diagnostic tool.

Humans↗
PubMed2005

Isothermal DNA amplification coupled with DNA nanosphere-based colorimetric detection.

We present a simple, rapid method for detecting short DNA sequences that combines a novel isothermal amplification method (EXPAR) with visual, colorimetric readout based on aggregation of DNA-functionalized gold nanospheres. The reaction is initiated by a trigger oligonucleotide, synthetic in nature for this proof-of-principle study, which is exponentially amplified at 55 degrees C and converted to a universal reporter oligonucleotide capable of bridging two sets of DNA-functionalized gold nanospheres. This reaction provides >10(6)-fold amplification/conversion in under 5 min. When combined with a solution containing DNA nanospheres, the bridging reporter causes nanosphere aggregation. The resulting color change from red to dark purple or blue is enhanced through spotting the solution onto a C18 reversed-phase thin-layer chromatography plate. The reaction can easily be adapted for detection of different trigger oligonucleotides using the same set of DNA nanospheres. It permits detection of as low as 100 fM trigger oligonucleotide in under 10 min total assay time, with minimal reagent consumption and requirement for instrumentation. We expect that combining this simple, versatile assay with trigger generation from a genomic target DNA sequence of interest will be a powerful tool in the development of rapid and simple point-of-care molecular diagnostic applications.

Base Sequence↗