LAMP-based Assay Research Articles

Development of loop-mediated isothermal amplification for detection of Leifsonia xyli subsp. xyli in sugarcane.

Ratoon stunt, caused by the xylem-limited coryneform bacterium Leifsonia xyli subsp. xyli (Lxx), is a deep bacteriosis and prevalent in most of sugarcane-producing countries. Based on loop-mediated isothermal amplification (LAMP), we developed a method for detecting Lxx. The major advantages of the LAMP method are visual judgment by color and time saving with only 60 min for identification of Lxx and without the need for costly PCR apparatus and gel scanner. In the present study, positive and negative samples detected by the LAMP method were clearly distinguishable. When total DNA extracted from internode juice was used as the template, the sensitivity of LAMP was 10 times higher than that of the conventional PCR detection. The LAMP assay is a highly specific, rapid, and sensitive method for the diagnosis of ratoon stunt caused by Lxx in sugarcane. This is the first report of LAMP-based assay for the detection of Lxx in sugarcane.

Development of loop-mediated isothermal amplification method for visualization detection of the highly virulent strains of porcine reproductive and respiratory syndrome virus (PRRSV) in China

A novel assay method to detect the highly virulent Porcine reproductive and respiratory syndrome virus (PRRSV) termed reverse transcriptase loop-mediated isothermal amplification (RT-LAMP), was reported by using hydroxynaphthol blue (HNB) as the LAMP product colorimetric judgment. By the set of special primers, targeting the sequence that belongs to Nsp2 gene containing an 87 bp deletion mutation, the LAMP-based assay could be completed within 1 h at 63°C. Final concentration of 150 mM HNB was confirmed to be appropriate in LAMP product judgment without impact on the reaction system. The detection limit of the assay was 103 copies per reaction, as determined by using a recombined plasmid. Genomes of various viruses were used to confirm the specificity of the LAMP-based assay, but only the highly virulent strains of PRRSV could be detected. All the internal organs from three stillborn piglets from a herd of pigs with PRRS-like symptoms were confirmed by LAMP-based assay to be infected not only with classical PRRSVs, but also the variant strains. These results suggest that the RT-LAMP assay with HNB provides a useful tool for the diagnosis of the highly virulent PRRSV infections in porcine rapidly. Key words: Reverse transcriptase loop-mediated isothermal amplification (RT-LAMP), highly virulent porcine reproductive and respiratory syndrome virus (PRRSV), classical porcine reproductive and respiratory syndrome virus (PRRSV), hydroxynaphthol blue (HNB).

Loop-mediated isothermal amplification for rapid detection of Chinese sacbrood virus

Chinese sacbrood virus (CSBV) has emerged as an important etiologic agent of honeybee infections and is lethal for individual bees, even causing the collapse of entire colonies. Although diagnostic methods for CSBV have been established in many clinical laboratories, application of these methods is largely restricted by the apparatus needed to carry out the reaction and by cost, therefore a simpler and less expensive diagnostic method for CSBV infection is required. In this study a simple and inexpensive system is described that is based on the loop-mediated isothermal amplification (LAMP) assay. The LAMP and the polymerase chain reaction (PCR) methods were compared for their ability to detect CSBV in 31 clinical samples, in purified CSBV-LNQY strains or to be able to discriminate between cDNA samples from other viruses. The detection limit of the LAMP method was 1 pg, showing that LAMP is as sensitive as reverse transcriptase (RT)-PCR for CSBV detection. In addition, no DNA band from other related viruses samples was amplified by either method, suggesting that this LAMP assay is as specific as RT-PCR for CSBV detection. All 31 clinical samples that were LAMP assay-positive were also amplified by RT-PCR, however the LAMP assay was faster, more cost effective, and easier to perform as the target gene amplified rapidly, within 2 h, and only a standard laboratory water bath or heat block was required for the reaction. The results demonstrate clearly that this LAMP-based assay is a useful tool for the rapid and sensitive diagnosis of CSBV infection of bees.

Rapid detection of the cariogenic pathogens Streptococcus mutans and Streptococcus sobrinus using loop‐mediated isothermal amplification

Streptococcus mutans and Streptococcus sobrinus are associated with the development of dental caries in humans. In this study, we developed a rapid, sensitive method for detecting these major cariogenic pathogens using loop-mediated isothermal amplification (LAMP). The assay procedure is quite simple: the amplification is carried out in a single tube under isothermal conditions at 63 degrees C, and the result can be obtained in less than 1 h. Initially, a set of six primers was designed by targeting S. mutans-specific and S. sobrinus-specific regions, identified using the genomic subtractive hybridization technique. We evaluated the specificities and sensitivities of these assays. Furthermore, we detected and quantified these bacteria in saliva and carious dentin from eight children. The sensitivities of the S. mutans-specific and S. sobrinus-specific LAMP methods, examined using agarose gel electrophoresis, were each one cell for a 30-min reaction. The detection limits using real-time turbidimetry analysis were 1 to 10(7) cells (3.28 x 10(1) to 3.28 x 10(8) fg S. mutans template DNA) per reaction tube and 1 to 10(5) cells (2.72 x 10(3) to 2.72 x 10(8) fg S. sobrinus template DNA) per reaction tube. Using these assays, we detected and quantified these cariogenic bacteria for evaluation of the LAMP assay for clinical diagnosis. Our results suggest that the LAMP-based assay in combination with subtractive hybridization is valuable for preparing species-specific primers for closely related species. Furthermore, the LAMP-based assay will be a useful tool for the rapid and sensitive prediction of dental caries.

Rapid detection of Actinobacillus actinomycetemcomitans using a loop‐mediated isothermal amplification method

Actinobacillus actinomycetemcomitans has been implicated in the etiology of aggressive periodontitis. In this study, we applied a novel nucleic acid amplification method, called loop-mediated isothermal amplification (LAMP), which amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions, allowing the rapid detection of A. actinomycetemcomitans. We designed the primers for detecting A. actinomycetemcomitans and evaluated the specificity and sensitivity of the assay. The LAMP primers used in this study successfully amplified serotypes a-e of A. actinomycetemcomitans, while other oral bacteria were not amplified. By measuring the precipitation of magnesium pyrophosphate, we could quantify the chromosomal DNA of A. actinomycetemcomitans. The detection limits using the real-time turbidimetry analysis were 5.8 x 10(2)-5.8 x 10(7) copies of A. actinomycetemcomitans template DNA per reaction tube. In addition, the LAMP assay was used for the rapid detection of A. actinomycetemcomitans in clinical specimens from eight individuals. The results with the LAMP method were similar to those using conventional polymerase chain reaction. Our results suggest that the LAMP-based assay is very useful for the rapid detection of A. actinomycetemcomitans.

Establishing a method of loop-mediated isothermal amplification for detecting resistant gene mecA rapidly in clinic sample

目的 建立环介导的等温扩增快速检测葡萄球菌耐药基因mecA的方法.方法利用包被有SPA单克隆抗体的磁珠富集样品中的金黄色葡萄球菌,快速提取其DNA, 用环介导的等温扩增反应(LAMP)扩增金黄色葡萄球菌耐药基因mecA,扩增条件为65°C 保温1 h.在反应过程中,荧光探针与mecA基因的扩增产物互补序列结合,产生荧光,通过检测反应管中的荧光值来判定结果.结果该方法的最低检测限为10 CFU/ml,与M-H培养基苯唑西林药敏试验结果相比较,灵敏性99%,特异性90.9%;而与PCR方法比较,灵敏性100%,特异性100%;试验时间缩短为1 h.结论该方法具有灵敏度高,特异性强,操作简便快速,适用于检测临床标本中的MRSA。

Loop-Mediated Isothermal Amplification for Rapid Detection of Newcastle Disease Virus

We have evaluated a diagnostic system based on the loop-mediated isothermal amplification (LAMP) assay for the rapid, simple, and sensitive detection of Newcastle disease virus (NDV) directly from culture isolates as well as clinical samples. By using one set of specific primers targeting the fusion protein gene, the LAMP assay rapidly amplified the target gene within 2 h, requiring only a regular laboratory water bath or heat block for reaction. The results obtained from testing the genomes of 38 NDV strains, other different viruses, and clinical samples of experimentally infected chickens showed that LAMP was as sensitive and specific as nested PCR. All LAMP-positive samples were positive by nested PCR. The LAMP assay is faster than nested PCR, cost-effective, and easy to perform. Our results clearly demonstrate that the LAMP-based assay is a useful tool for the rapid and sensitive diagnosis of NDV infection.