Detection of Mycoplasma pneumoniae by loop-mediated isothermal amplification (LAMP) assay and serology in pediatric community-acquired pneumonia

Rapid diagnosis of Mycoplasma pneumoniae pneumonia is required for treatment with effective antimicrobial agents without delay; however, this capacity has not yet been established in clinical practice. Recently, a novel nucleic acid amplification method termed loop-mediated isothermal amplification (LAMP) has been used to rapidly diagnose various infectious diseases. In this study, we prospectively evaluated the efficacy of the LAMP assay to rapidly diagnose M. pneumoniae pneumonia in clinical practice. Three hundred sixty-eight children (median age, 3.8 years; range, 0.1–14.3 years) admitted to our hospital between April 2009 and March 2010 for community-acquired pneumonia were enrolled in this study. We obtained throat swabs on admission to detect M. pneumoniae DNA and paired serum samples on admission and at discharge to assay M. pneumoniae antibody titers. M. pneumoniae pneumonia was diagnosed by either a positive LAMP assay or a fourfold or greater increase in antibody titer. Overall, 46 children (12.5% of the patients with pneumonia) were diagnosed with M. pneumoniae pneumonia; of these, 27 (58.7%) were aged less than 6 years. Of the aforementioned 46 children, 38 (82.6%) and 37 (80.4%) were identified by LAMP and serology, respectively. When the results of serology were taken as the standard, the sensitivity and specificity and positive and negative predictive values of the LAMP assay were 78.4%, 97.3%, 76.3%, and 97.6%, respectively. We concluded the LAMP assay may be useful for rapid diagnosis of M. pneumoniae pneumonia.     

Simultaneous diagnosis of dengue virus,Chikungunya virus,and Zika virus infection using a new point-of-care testing (POCT) system based on the loop-mediated isothermal amplification (LAMP) method

IntroductionDengue fever, Chikungunya fever, and Zika virus infection have similar symptoms and overlapping areas of endemicity and so cannot be distinguished clinically. Rapid diagnosis tests are available for each infection but each test covers only single target. There are PCR-based methods available that test for all three infections simultaneously, not applicable for in-vitro diagnostic use. The aim of this study was to evaluate the diagnostic accuracy of a new point-of-care testing (LAMP POCT) based on loop-mediated isothermal amplification method that tests for all three viruses simultaneously, using serum or urine samples, and takes about 45 min.MethodsLAMP POCT was evaluated as the comparator on 67 individuals, of whom 56 had dengue, three had Chikungunya, two had Zika virus infection and six did not have any kind of these infections confirmed by RT-PCR.ResultsAmong individuals with dengue, the sensitivity of LAMP POCT was 100% in samples collected in the first 3 days after illness onset, all three patients with Chikungunya were LAMP POCT positive, and both patients with Zika virus infection were LAMP POCT negative. There were no false-positive test results.ConclusionsLAMP POCT has potential utility as a point-of-care combination test for dengue and Chikungunya viruses, but further prospective studies are needed among individuals with Chikungunya virus and Zika virus infection, using serum and urine samples.     

E.coli O157：H7 LAMP检测方法的建立

目的建立检测E.coli O157：H7环介导的等温扩增方法（LAMP）。方法选取E.coli O157：H7的rfbE基因设计LAMP引物,优化建立LAMP检测体系。并用该体系和国标法对76份人工污染样品进行了检测。结果所建立的E.coli O157：H7 LAMP体系具有良好的特异性,用该体系对9属13种41株菌进行检测,结果只有E.coli O157：H7的扩增产物电泳结果为阶梯状条带,非E.coli O157：H7均未产生扩增产物,灵敏性为2.7×101CFU/mL。样品及培养基对反应体系没有影响或影响很小。对76份样品进行了检测,E.coli O157：H7 LAMP检测方法与国标法的总符合率为96.1%,2h内即可完成检测。结论本研究建立的E.coli O157：H7的LAMP法不但解决了分子生物学检测对实验室仪器设备的高要求问题,而且检测方法简便、快捷,非常便于基层实验室和现场检测使用。     

Specific detection of Angiostrongylus cantonensis in the snail Achatina fulica using a loop-mediated isothermal amplification (LAMP) assay

Angiostrongylus cantonensis, a rat lungworm, can cause eosinophilic meningitis and angiostrongyliasis in humans following ingestion of contaminated foods or intermediate/paratenic hosts with infective larvae. The snail Achatina fulica is one of the important intermediate hosts of A. cantonensis and is commonly eaten by humans in some countries. In the present study, we developed a loop-mediated isothermal amplification (LAMP) method for the specific detection of A. cantonensis in Ac. fulica. Primers for LAMP were designed based on the first internal transcribed spacer (ITS-1) of nuclear ribosomal DNA (rDNA) of A. cantonensis. Specificity tests showed that only the products of A. cantonensis were detected when DNA samples of A. cantonensis and the heterologous control samples Anisakis simplex s.s, Trichuris trichiura, Toxocara canis, Trichinella spiralis and Ascaris lumbricoides were amplified by LAMP. Sensitivity evaluation indicated that the LAMP assay is 10 times more sensitive than the conventional polymerase chain reaction (PCR) assay. The established LAMP assay is rapid, inexpensive and easy to be performed. It can be used in clinical applications for rapid and sensitive detection of A. cantonensis in snails, which has implications for the effective control of angiostrongyliasis.     

基于环介导恒温扩增法快速检测破伤风梭菌

目的 利用环介导恒温扩增技术,设计快速检测破伤风梭菌的方法,以便对破伤风梭菌感染进行快速检测.方法 (1)设计针对破伤风梭菌的环介导恒温扩增检测方法.(2)对破伤风梭菌恒温扩增法进行特异性试验.(3)对破伤风梭菌恒温扩增法进行灵敏度试验.结果 (1)设计出针对破伤风梭菌的恒温扩增检测方法.(2)本恒温扩增检测方法只扩增...     

高灵敏度等温扩增法可视化检测血液中的病毒核酸

目的探讨便携式、可灵敏、快速检测血液标本中病毒核酸的方法。方法以HBV病毒为例,针对其基因组保守序列设计特异性引物,采用环介导等温扩增法(LAMP)对待测HBV标本做核酸目标序列扩增,以高灵敏双链DNA嵌合荧光染料为指示剂,根据荧光信号指示扩增反应产物的有无;将本法与实时荧光PCR法对临床标本做HBV检测的对照分析。结果建立了血液病毒核酸的可视化检测方法,研制出扩增检测一体化仪器;灵敏度达到可在<1 h完成10 cp/管的HBV核酸扩增反应和产物检测。62例实时荧光PCR检测为HBV阳性的临床标本,本法亦都检测为阳性;而从42例实时荧光PCR检测为HBV阴性的临床标本中,本法检出了6例HBV阳性。结论所建立的方法和装置可实现高灵敏度单管快速检测血液病毒核酸,为特殊现场环境中的血液安全性筛查提供了新的手段。     

副溶血弧菌的gyrB基因环介导的恒温扩增技术检测方法的研究

目的建立一种适合基层实验室应用和开展的快速检测副溶血弧菌的DNA环介导的恒温扩增法(LAMP)。方法针对副溶血弧菌的IgyrB/I 基因序列设计了4条引物（2条内引物、2条外引物）,并对扩增反应条件进行了优化。结果整个检测过程仅需1.5 h,可通过肉眼目测或电泳检 测判断结果;对3株种系背景明确的副溶血弧菌不同实验对照株、23株副溶血弧菌地方分离株和32株其他肠道菌进行了检测,具有很高的特异性 ;该方法的最低检测限为24 cfu/ml,具有良好的敏感性;应用于61份贝类海产品的现场检测,阳性率为100%,与实时荧光定量PCR方法的检测 结果相符,阳性率高于传统的培养鉴定方法。结论本方法具有快速、灵敏、特异、简便、经济等特点,适合基层实验室、应急检测或现场监测 等使用,具有较高的推广价值。     

Rapid diagnosis of largemouth bass ranavirus in fish samples using the loop-mediated isothermal amplification method

Largemouth bass ranavirus (LMBV) has been recognized as the causative pathogen responsible for infectious skin ulcerative syndrome in cultured largemouth bass in China. A fast and simple LMBV detection method is urgently needed. Here, a loop-mediated isothermal amplification (LAMP) assay was established for the detection of this virus using primers targeting the major capsid protein gene of LMBV. The amplification conditions were optimized; the assay was specific for the diagnosis of LMBV, as there was no cross-reactivity with other four Iridoviridae viruses (large yellow croaker iridovirus, Singapore grouper iridovirus, tiger frog virus, and soft-shelled turtle iridovirus), grass carp reovirus, white spot syndrome virus, or healthy largemouth bass. The sensitivity of the LAMP assay was found to be 8.55 × 10¹ copies/μL of LMBV DNA, which was 10-fold higher than that of the conventional PCR. Application of the LAMP assay was evaluated using 10 clinical samples, and the results indicated the reliability of the test as a rapid, field diagnostic tool for LMBV detection. Thus, the simplicity and nearly instrument-free LAMP method provides an alternative for rapid and sensitive detection of LMBV and has great potential for early diagnosis of LMBV infection in the farm.     

Loop-mediated isothermal amplification (LAMP): recent progress in research and development

Loop-mediated isothermal amplification (LAMP) is an established technology that continues to attract the attention of researchers in many fields. Research and development efforts on LAMP technology in recent years have focused on two major areas; first, the study of its clinical application as an approved in vitro diagnostics tool in Japan and certain other countries; and second, research aimed at further simplifying the LAMP test process. This review provides an overview of the status of LAMP on these two topics by summarizing research work conducted, in the main, after our previous review article.     

Rapid and sensitive detection of human African trypanosomiasis by loop-mediated isothermal amplification combined with a lateral-flow dipstick

A combined loop-mediated isothermal amplification lateral flow dipstick (LAMP-LFD) format was evaluated in the detection of human infective trypanosome DNA from clinical samples. The LAMP-LFD showed analytical sensitivity equivalent to 0.01 tryps/mL, levels that were identical to using gel electrophoresis and SYBR Green I dye. The LAMP-LFD showed superior specificity to SYBR Green I when supernatant prepared from boiled human biological samples was used as template. These results indicate that the use of nonspecific DNA intercalators may produce false positives when partially processed templates are used. The LAMP-LFD format presented here is simple, rapid, and has future potential use in diagnosis of sleeping sickness.     

乙型脑炎和登革热病毒的环介导等温扩增基因定量检测技术研究

目的建立一种适合口岸现场快速检测乙型脑炎和登革热病毒的环介导等温扩增(LAMP)定量技术。方法根据LAMP方法的原理,设计LAMP检测引物和反应体系,建立LAMP检测方法,同时综合评估初始拷贝数值与方法中的灵敏度、特异度、重复性及荧光信号值反应时间(1×104)之间的线性关联。结果检测选用1套LAMP引物,完成时间为0.5h,传统PCR检测与LAMP对比,差异明显,LAMP检测可有效提高灵敏度,是PCR检测技术的10倍。循环阈值和模版浓度具有良好的线性联系,实验室变异系数为小于5%。结论该方法是特异度与灵敏度较高,操作简单、结果判断容易、设备要求低的快速检测方法,适合基层医疗卫生机构和现场查验机构的广泛应用。     

A successful diagnostic case of Pneumocystis pneumonia by the loop-mediated isothermal amplification method in a patient with dermatomyositis

Pneumocystis pneumonia (PCP) can occur in patients with many causes of the immunocompromised state other than human immunodeficiency virus (HIV). It is quite difficult to diagnose PCP without HIV because there is no method for detecting Pneumocystis jirovecii. Thus, non-HIV PCP continues to have high mortality. Recently, loop-mediated isothermal amplification (LAMP) is becoming an established nucleic acid amplification method offering rapid, accurate, and cost-effective diagnosis of infectious diseases. We report a non-HIV PCP case successfully diagnosed by the LAMP method. It was previously reported that PCR in BALF specimens had been the most sensitive method in the diagnosis of PCP without HIV. The LAMP method would be more sensitive than conventional PCR and an effective tool in the early diagnosis of PCP.     

环介导等温扩增技术快速检测结核分枝杆菌核酸

摘要:目的：建立一种快速准确的检测结核分枝杆菌（MTB）核酸的环介导等温扩增（LAMP）方法。 方法：以重复插入序列IS6110为目的基因,设计LAMP引物,特异检测MTB核酸。用本法与痰涂片抗酸染色镜检法、实时荧光PCR法对100例可疑患者痰标本进行对比检查。 结果：LAMP法特异性强,仅扩增MTB复合群核酸;灵敏度高,检测限达100 fg;而实时荧光PCR检测限为1 pg。对100例疑似结核病患者痰液标本检测,涂片抗酸染色法、LAMP法、实时荧光PCR法的阳性率分别为28%、39%和38%。 结论：本研究建立的LAMP方法检测MTB核酸特异性强、灵敏度高、时间短且操作简便,有望成为临床快速检测MTB的新方法。     

Sensitive and rapid detection of Aeromonas caviae in stool samples by loop-mediated isothermal amplification

In this study, a loop-mediated isothermal amplification assay targeting the 16S-23S intergenic spacer regions (internal transcribed spacer) of Aeromonas caviae was developed. Eighteen reference strains and 109 clinical samples were analyzed. The results showed this detection technique is more reliable and convenient compared with common polymerase chain reaction and biochemical culture methods.     

环介导的等温扩增技术对HBV、HCV和HIV核酸检测的研究

目的建立环介导的等温扩增技术(LAMP)对HBV、HCV和H IV核酸检测的方法并对检测灵敏度和特异性作初步考核。方法通过引物设计和筛选、内质控的设计与时间分辨浊度检测方法的运用,建立LAMP HBVDNA、HCV RNA和H IV RNA扩增体系;用连续稀释的阳性样本和不含任何病毒核酸的正常人血浆考核所建立的LAMP扩增体系的灵敏度和特异性。结果建立了LAMP HBV DNA扩增体系及HCV/H IV RNA双联检体系,该体系对5 CP/m l的HBV DNA样本的检出率为51.85%,对100 CP/m l的HCV RNA阳性样本的检出率为61.90%,对100 CP/m l的H IV RNA阳性样本的检出率为45%。对考核样本检测的相对灵敏度和特异性均为100%。结论LAMP HBV、HCV和H IV检测灵敏度较高,在进一步优化以后能用于HBV、HCV和H IV的核酸检测。     

Application of a loop-mediated isothermal amplification method for the detection of pathogenic Leptospira

Leptospirosis is an emerging infectious disease, which is considered to be the most widespread zoonotic disease in the world. There are more than 230 known serovars in the genus Leptospira. A loop-mediated isothermal amplification (LAMP) assay for the rapid detection of pathogenic Leptospira spp. was developed and evaluated through amplification of the lipL41 gene coding for the outer membrane protein LipL41. The LAMP assay did not rely on the isolation and culture of leptospires, and no cross-reactivity was observed with other bacterial species. A SYBR Green I-based LAMP assay was also carried out for the real-time detection of DNA amplification. The lower detection limit of the LAMP assay was approximately 100 copies, which was the same as the polymerase chain reaction (PCR) and real-time PCR assays. The accuracy of the LAMP reaction was confirmed by restriction endonuclease analysis of the amplified product. The LAMP assay is easy to perform and inexpensive, and so may be applied in the rapid and specific diagnosis of Leptospira.     

Application of loop-mediated isothermal amplification technique to rapid and direct detection of methicillin-resistant Staphylococcus aureus (MRSA) in blood cultures

Staphylococcus aureus is the most important pathogen in nosocomial infections, including bloodstream infections. Prompt identification of S. aureus from blood cultures and detection of methicillin resistance are essential in cases of suspected sepsis. A novel nucleic acid amplification technique, loop-mediated isothermal amplification (LAMP), which amplifies DNA under isothermal conditions (63°C) with high specificity, efficiency, and rapidity, was applied to detect methicillin-resistant S. aureus (MRSA) directly from positive blood culture bottles. MRSA-LAMP, which targets the spa gene, encoding S. aureus-specific protein A, and the mecA gene, encoding penicillin-binding protein-2′ for methicillin resistance, could detect MRSA within 2 h after the blood culture signal became positive. The diagnostic values of LAMP, compared to a duplex real-time polymerase chain reaction (Drt-PCR) assay, were 92.3% and 96.2% sensitivity, 100% and 100% specificity, 100% and 100% positive predictive value (PPV), and 96.9% and 98.4% negative predictive value (NPV), respectively. These two methods had almost the same results, but the LAMP method is more cost-effective and provides excellent availability for rapid examination in a hospital clinical laboratory. Therefore, the LAMP assay appears to be a sensitive and reliable new method to diagnose MRSA bloodstream infection for appropriate antibiotic therapy.     

Comparison of loop-mediated isothermal amplification (LAMP) and nested-PCR assay targeting the RE and B1 gene for detection of Toxoplasma gondii in blood samples of children with leukaemia

Toxoplasmosis diagnosis constitutes an important measure for disease prevention and control. In this paper, a newly described DNA amplification technique, loop-mediated isothermal amplification (LAMP), and nested-PCR targeting the repeated element (RE) and B1 gene, were compared to each other for the detection of Toxoplasma gondii DNA in blood samples of children with leukaemia. One hundred ten blood samples from these patients were analyzed by LAMP and nested-PCR. Out of 50 seropositive samples (IgM+, IgG+), positive results were obtained with 92% and 86% on RE, B1-LAMP and 82% and 68% on RE, B1-nested PCR analyses, respectively. Of the 50 seronegative samples, three, two and one samples were detected positive by RE-LAMP, B1-LAMP and RE-nested PCR assays, respectively, while none were detected positive by B1-nested PCR. None of the 10 IgM−, IgG+ samples was detected positive after testing LAMP and nested-PCR assays in duplicate. This is the first report of a study in which the LAMP method was applied with high sensitivity and efficacy for the diagnosis of T. gonii in blood samples of children with leukaemia.     

Rapid and sensitive detection of Vibrio vulnificus by loop-mediated isothermal amplification combined with lateral flow dipstick targeted to rpoS gene

A novel loop-mediated isothermal amplification (LAMP) combined with amplicon detection by chromatographic lateral flow dipstick (LFD) assay was developed and evaluated for the detection of Vibrio vulnificus. Biotinylated LAMP amplicons were produced by a set of six designed primers that recognized the V. vulnificus RNA polymerase subunit sigma factor S (rpoS) gene followed by hybridization with an FITC-labeled probe and LFD detection. The optimized time and temperature conditions for the LAMP assay were 90 min at 65 °C. The LAMP–LFD method accurately identified 14 isolates of V. vulnificus but did not detect 25 non-vulnificus Vibrio isolates and 37 non-Vibrio isolates. The sensitivity of LAMP–LFD for V. vulnificus detection in pure culture was 1.5 × 10³ CFU ml⁻¹ or equivalent to 2.8 CFU per reaction. In the case of spiked oyster samples without enrichment, the detection limit for V. vulnificus was 1.2 × 10⁴ CFU g⁻¹ or equivalent to 11 CFU per reaction. The results show that this method appears to be accurate, precise and valuable tool for identification of V. vulnificus and can be used efficiently for detection of V. vulnificus in contaminated food sample.     

Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases

Loop-mediated isothermal amplification (LAMP) is an established nucleic acid amplification method offering rapid, accurate, and cost-effective diagnosis of infectious diseases. This technology has been developed into commercially available detection kits for a variety of pathogens including bacteria and viruses. The current focus on LAMP methodology is as a diagnostic system to be employed in resource-limited laboratories in developing countries, where many fatal tropical diseases are endemic. The combination of LAMP and novel microfluidic technologies such as Lab-on-a-chip may facilitate the realization of genetic point-of-care testing systems to be used by both developed and developing countries in the near future. This review will describe the historical, current, and future developments of such technologies.