Simultaneous multiple target detection in real-time loop-mediated isothermal amplification

Article (PDF Available)inBioTechniques 53(2):81-9 · August 2012with860 Reads
DOI: 10.2144/0000113902 · Source: PubMed
Loop-mediated isothermal amplification (LAMP) is a rapid and reliable sequence-specific isothermal nucleic acid amplification technique. To date, all reported real-time detection methods for LAMP have been restricted to single targets, limiting the utility of this technique. Here, we adapted standard LAMP primers to contain a quencher-fluorophore duplex region that upon strand separation results in a gain of fluorescent signal. This approach permitted the real-time detection of 1-4 target sequences in a single LAMP reaction tube utilizing a standard real-time fluorimeter. The methodology was highly reproducible and sensitive, detecting below 100 copies of human genomic DNA. It was also robust, with a 7-order of magnitude dynamic range of detectable targets. Furthermore, using a new strand-displacing DNA polymerase or its warm-start version, Bst 2.0 or Bst 2.0 WarmStart DNA polymerases, resulted in 50% faster amplification signals than wild-type Bst DNA polymerase, large fragment in this new multiplex LAMP procedure. The coupling of this new multiplex technique with next generation isothermal DNA polymerases should increase the utility of the LAMP method for molecular diagnostics.

Full-text (PDF)

Available from: Yinhua Zhang, Dec 21, 2013
    • "The limit of detection of the LAMP assay resulted tenfold higher when Bst polymerase 2.0 WarmStart was used in comparison with Bst polymerase 2.0 (corresponding to 0.01 ng vs. 0.1 ng, respectively). It has been previously reported a number of advantages of Bst polymerase WarmStart version compared to other commercially available versions, such as faster amplification [54], increased stability at room temperature [55] and also greater sensitivity [56, 57]. We emphasize the importance of setting up the best conditions and molecular components for primers set operation in a LAMP assay. "
    [Show abstract] [Hide abstract] ABSTRACT: Background: Strongyloides stercoralis, the chief causative agent of human strongyloidiasis, is a nematode globally distributed but mainly endemic in tropical and subtropical regions. Chronic infection is often clinically asymptomatic but it can result in severe hyperinfection syndrome or disseminated strongyloidiasis in immunocompromised patients. There is a great diversity of techniques used in diagnosing the disease, but definitive diagnosis is accomplished by parasitological examination of stool samples for morphological identification of parasite. Until now, no molecular method has been tested in urine samples as an alternative to stool samples for diagnosing strongyloidiasis. This study aimed to evaluate the use of a new molecular LAMP assay in a well-established Wistar rat experimental infection model using both stool and, for the first time, urine samples. The LAMP assay was also clinically evaluated in patients´ stool samples. Methodology/principal findings: Stool and urine samples were obtained daily during a 28-day period from rats infected subcutaneously with different infective third-stage larvae doses of S. venezuelensis. The dynamics of parasite infection was determined by daily counting the number of eggs per gram of feces from day 1 to 28 post-infection. A set of primers for LAMP assay based on a DNA partial sequence in the 18S rRNA gene from S. venezuelensis was designed. The set up LAMP assay (namely, Strong-LAMP) allowed the sensitive detection of S. venezuelensis DNA in both stool and urine samples obtained from each infection group of rats and was also effective in S. stercoralis DNA amplification in patients´ stool samples with previously confirmed strongyloidiasis by parasitological and real-time PCR tests. Conclusions/significance: Our Strong-LAMP assay is an useful molecular tool in research of a strongyloidiasis experimental infection model in both stool and urine samples. After further validation, the Strong-LAMP could also be potentially applied for effective diagnosis of strongyloidiasis in a clinical setting.
    Full-text · Article · Jul 2016
    • "It was highly reduced through the use of equimolar standard FIP primer and probe. It may be due to faster target generation with FIP and easier incorporation of duplex FIP during exponential amplification , and using equimolar amounts maintains rapid threshold detection with moderate fluorescence signal amplitude (Tanner et al., 2012). In addition to amplification curve, positive results would be further confirmed by turbidity and addition of SYBR green at the end of reaction. "
    [Show abstract] [Hide abstract] ABSTRACT: A one step, single tube, accelerated probe based real time loop mediated isothermal amplification (RT LAMP) assay was developed for detecting the invasion gene (InvA) of Salmonella. The probe based RT LAMP is a novel method of gene amplification that amplifies nucleic acid with high specificity and rapidity under isothermal conditions with a set of six primers. The whole procedure is very simple and rapid, and amplification can be obtained in 20min. Detection of gene amplification was accomplished by amplification curve, turbidity and addition of DNA binding dye at the end of the reaction results in colour difference and can be visualized under normal day light and in UV. The sensitivity of developed assay was found 10 fold higher than taqman based qPCR. The specificity of the RT LAMP assay was validated by the absence of any cross reaction with other members of enterobacteriaceae family and other gram negative bacteria. These results indicate that the probe based RT LAMP assay is extremely rapid, cost effective, highly specific and sensitivity and has potential usefulness for rapid Salmonella surveillance.
    Article · Apr 2016
    • "Real-time LAMP reactions using primer set 2 can use either of the real-time protocols mentioned previously, containing either 5 µl sample (or DNA), 5 µl primer master mix [ DNase/RNase-free H 2 O). The primers that use this alternate probe design are more expensive, as more fluorescent probe is used with each reaction; however the benefit comes for its ability to be successfully multiplexed (Tanner et al., 2012). The new probe design was applied to a LAMP assay designed for specific detection of Clavibacter michiganensis subsp. "
    [Show abstract] [Hide abstract] ABSTRACT: Potatoes are an important agroeconomic crop worldwide and maceration diseases caused by pectolytic bacterial pathogens result in significant pre- and postharvest losses. Pectobacterium carotovorum shares a common host range with other Pectobacterium spp. and other members of the Enterobacteriaceae such as Dickeya spp. As these pathogens cannot be clearly differentiated based on the symptoms they cause, improved methods of identification are critical for determining sources of contamination. Current standardized methods for differentiation of pectolytic species are time consuming and require trained personnel, as they rely on traditional bacteriological practices that do not always produce conclusive results. In this growing world market, there is a need for rapid diagnostic tests that can differentiate between pectolytic pathogens, as well as separate them from nonpectolytic enteric bacteria associated with soft rots of potato. An assay has been designed previously to detect the temperate pathogen P. atrosepticum, but there is currently no recognized rapid assay for detection of the tropical/subtropical counterpart, P. carotovorum. This report describes the development of a loop-mediated isothermal amplification (LAMP) assay that detects Pectobacterium carotovorum with high specificity. The assay was evaluated using all known species of Pectobacterium and only showed positive reactions for P. carotovorum. This assay was also tested against 15 non-target genera of plant-associated bacteria and did not produce any false positives. The LAMP assay described here can be used as a rapid test for differentiating Pectobacterium carotovorum from other pectolytic pathogens, and its gene target can be the basis for development of other molecular-based detection assays. This article is protected by copyright. All rights reserved.
    Full-text · Article · Feb 2016
Show more