Evaluation of a loop-mediated isothermal amplification assay for rapid and simple detection of Vibrio parahaemolyticus in naturally contaminated seafood samples

We investigated the efficacy of a loop-mediated isothermal amplification (LAMP) assay for rapid screening of seafood samples naturally contaminated with Vibrio parahaemolyticus. A total of 171 seafood samples enriched in alkaline peptone water (APW) were assessed by LAMP assay and conventional culture methods, which consist of a combination of APW enrichment culture and plating onto CHROMagar Vibrio and TCBS agars. Compared with V. parahaemolyticus isolation using the conventional culture test, LAMP results showed 100% (30/30) and 90.8% (128/141) sensitivity and specificity, respectively. The conventional culture test required more than 3 days to isolate and identify V. parahaemolyticus in the APW enrichment culture. In contrast, the LAMP assay was markedly faster, requiring less than 60 min from the beginning of DNA extraction to final detection of V. parahaemolyticus. In total, the LAMP assay required 17-19 h from the beginning of enrichment culture to final determination. This is the first report of the LAMP assay for rapid screening of seafood samples naturally contaminated by V. parahaemolyticus.     

Rapid detection of Vibrio parahaemolyticus strains and virulent factors by loop-mediated isothermal amplification assays

A loop-mediated isothermal amplification (LAMP) method for rapid detection of the foodborne Vibrio parahaemolyticus strains and related virulent factors had been developed and evaluated in this study. Six primers, including outer primers, inner primers, and loop primers, were specially designed for recognizing 8 distinct sequences on 3 target genes, which were tlh, tdh, and trh. The detection limits were found to be 100, 100 fg, and 1 pg DNA/tube for tlh, tdh, and trh, respectively. Application of LAMP assays were performed on 368 foodborne V. parahaemolyticus strains, the sensitivities of LAMP assays for the tlh, tdh, and trh were 100, 95.6, and 96.4%, and the negative predictive values (NPV) were 100, 84.7, and 93.1%, respectively; with a 100% specificity and positive predictive value (PPV) for all 3 target genes.     

金黄色葡萄球菌LAMP可视化快速检测方法的建立

建立金黄色葡萄球菌LAMP可视化检测方法。通过对2套引物的筛选,最终选取nuc基因的6条引物建立LAMP反应体系。整个反应在63℃恒温条件下进行45min,即可通过肉眼直接对反应结果进行判定。对金黄色葡萄球菌的检测灵敏度为0.001ng/μL,且与其他致病菌无交叉反应。该方法快速、特异性高,操作简便,适合基层快速检测。     

Development of a fimY-based loop-mediated isothermal amplification assay for detection of Salmonella in food

In this study, we developed a rapid and sensitive fimY-based loop-mediated isothermal amplification (LAMP) assay on a real-time turbidimeter platform for the detection of Salmonella in food. Since turbidity of the reaction mixture would increase in correlation with the DNA yield, real-time monitoring of the LAMP reaction was achieved by real-time turbidimeter. Time threshold values which indicate positive results for 81 Salmonella strains of different serotypes ranged from 36 to 40 min. For the 20 non-Salmonella strains, turbidity did not increase in the reaction mixture. When testing 10-fold serial dilutions of Salmonella Typhimurium-ATCC 14128 DNA by LAMP, the time threshold ranged from 36 to 52 min on the real-time turbidimeter. The detection limit was 13 cells per reaction in pure culture, up to 10-fold more sensitive than that of PCR. When applied in deli food samples, the LAMP assay was able to detect Salmonella even though the sample was contaminated with very low concentration after 3 h enrichment culture. Increase in turbidity was observed on real-time turbidimeter. Additionally, the LAMP results detected by naked-eye or turbidity consistently matched with each other. Results from this study showed that the fimY-based LAMP assay is an effective method for the rapid detection of Salmonella.     

环介导恒温扩增技术可视化检测携带tdh基因的致病性副溶血性弧菌

建立了环介导恒温扩增技术检测携带tdh基因的致病性副溶血性弧菌。基于副溶血性弧菌高度保守的tdh基因序列,设计了6条特异性引物,两条外引物F3、B3,两条内引物FIP、BIP及两条环引物LF、LB。在Bst DNA Polymerase作用下,60℃恒温水浴进行扩增。对10种细菌共21株菌进行LAMP扩增,所试6株副溶血性弧菌均为阳性,说明引物具有高度特异性。本LAMP方法对纯培养物的灵敏度可达到9.42cfu/m L。对污染食品中副溶血性弧菌的灵敏度为25.3cfu/25g,40~60min内即可完成检测。本方法操作简便、特异性强、灵敏度高,可以为临床提供简单、快速、高灵敏度和高特异性的检测应用。      

Development and application of a rapid and simple loop-mediated isothermal amplification method for food-borne Salmonella detection

Food Science and Biotechnology - A loop-mediated isothermal amplification (LAMP) method for rapid detection of the food-borne Salmonella strains had been developed and evaluated in this study. The...     

海产品中副溶血性弧菌免疫磁分离和环介导等温扩增快速检测方法的建立

采用纳米免疫磁珠分离副溶血性弧菌,建立副溶血性弧菌环介导等温扩增检测方法。方法 采用副溶血性弧菌单克隆抗体,制备纳米免疫磁珠,特异性吸附副溶血性弧菌,结合环介导等温扩增技术,建立副溶血性弧菌快速检测方法。结果 副溶血性弧菌纳米免疫磁珠在菌体浓度为10³cfu/ml水平时,对副溶血性弧菌的捕获率达到74%。免疫磁分离结合环介导等温扩增技术,在纯培养、无需增菌情况下,检测灵敏度达到140cfu/ml增菌液;通过对134株副溶血性弧菌和74株非目标菌的测试,环介导等温扩增技术具有良好的特异性;食品基质添加试验中,在增菌时间缩短至8h的条件下,其检测限为2cfu/25g样品。结论 副溶血性弧菌免疫纳米磁珠结合环介导等温扩增技术,有效缩短了增菌时间,适用于副溶血性弧菌的快速检测。     

环介导等温扩增技术结合乳胶微球试纸条检测副溶血性弧菌

目的 建立环介导等温扩增(loop-mediated isothermal amplification, LAMP)技术结合乳胶微球试纸条(latex microsphere test strips, LMTS)快速检测副溶血性弧菌(Vibrio parahaemolyticus, VP)的方法。方法 以副溶血性弧菌的不耐热溶血素(TLH)基因作靶标设计引物,6-羧基荧光素(6-carboxyfluorescein,6-FAM)和生物素(Biotin)标记引物,对体系各项反应参数进行优化;将新鲜菌液作10倍梯度稀释后进行LAMP、聚合酶链式反应(polymerase chain reaction, PCR)和实时荧光定量LAMP(quantitative LAMP, qLAMP)反应,比较三者的敏感度;对副溶血性弧菌、大肠埃希氏菌、福氏志贺氏菌、粪链(肠)球菌、单核细胞增生李斯特菌、鼠伤寒沙门氏菌、铜绿假单胞菌进行LAMP扩增,验证其特异性;虾样品用菌液进行模拟污染,分析LAMP-LMTS的可靠性。结果 LAMP-LMTS方法的灵敏度可达4.16×10² copi...     

Sensitive and rapid detection of Alicyclobacillus acidoterrestris using loop-mediated isothermal amplification

BACKGROUND: A loop‐mediated isothermal amplification (LAMP) assay was developed for the rapid detection (within 2 h) of Alicyclobacillus acidoterrestris. The assay detected the species‐specific DNA sequence of the 16S–23S rRNA internal transcribed spacer. RESULTS: The eight strains of A. acidoterrestris were successfully amplified, but six strains of other bacillus Acidocaldarius and 13 bacterial species other than bacillus Acidocaldarius were not. The sensitivity of the LAMP assay was at 4.50 × 10^?2 cfu per tube. This sensitivity is greater than that obtained by polymerase chain reaction (PCR) assay. The LAMP assay was examined further for its ability to detect A. acidoterrestris in juice samples. The results were compared with those of conventional PCR detection. CONCLUSION: Results indicate that the proposed LAMP assay is a rapid, specific and sensitive method for detecting A. acidoterrestris. As the amplification has been conducted under isothermal conditions, only a water bath or heating block is needed to maintain the required temperature. Thus, the method can be generalised and popularised easily in the future. Copyright © 2011 Society of Chemical Industry     

LAMP技术快速检测海产品副溶血弧菌的条件优化

本研究以副溶血弧菌不耐热溶血素tlh为目标基因设计特异性引物,优化反应条件,并对特异性、灵敏度进行了验证。LAMP最佳反应条件为,外内引物浓度比为1∶8,Mg2+反应浓度为4 mmol/L,d NTPs浓度为3.5 mmol/L,温度为65℃,反应时长为1 h,只对副溶血弧菌产生扩增反应,与其余细菌不产生扩增反应,细菌基因组DNA和纯培养物的灵敏度约为5.45×101 fg/μL和140 cfu/m L,对人工污染食品样品进行检测,检出限为2.8×102 cfu/m L。该方法反应时间短、特异性强、灵敏度高。      

水产品和水体中创伤弧菌现场可视化环介导恒温扩增快检方法的建立及应用

目的 基于环介导恒温扩增(LAMP)技术建立水产品和养殖水域中创伤弧菌现场可视化快速、简便的检测方法。方法 以创伤弧菌作为研究对象,分别采用煮沸法和离心柱法提取基因组DNA,优化简便、快速的弧菌DNA提取方法;以创伤弧菌gyrB基因作为靶基因,设计筛选最佳的适于LAMP的引物序列,分别通过LAMP荧光扩增曲线(加SYTO-9 荧光染料),以及LAMP可视化观察扩增产物的颜色变化(加钙黄绿素),开展特异性、灵敏度和重复性试验分析。结果 确定煮沸法为适合于弧菌基因组DNA提取的快捷方法;优化筛选的引物可以特异地检测创伤弧菌,检测核酸浓度的灵敏度可以达到10 fg/μL,并且结果稳定、可靠。采用该方法进行实际样品的检测应用,在558份水体样品和655份水产品样品中,创伤弧菌阳性检出率分别为9.01%和8.60%,阳性检出率高于传统的分离培养鉴定结果。结论 低技术要求、低设备成本以及检测时间短使得可视化LAMP快检方法成为现场可使用的一个理想选择,对于水产养殖业来说也更方便。     

LAMP实时浊度法检测转基因水稻Bt63品系

采用环介导等温扩增技术(LAMP),针对转基因水稻Bt63品系外源基因与内源基因结合处序列设计4条特异性引物,利用浊度仪实时监测扩增过程,建立Bt63品系的快速检测技术体系,并对反应温度、灵敏度、特异性、稳定性和实际样品检测能力进行初步探索。结果表明,LAMP实时浊度法能够特异性检测转基因水稻Bt63品系,其最低检出限为0.01%,是普通PCR方法的10倍,具有广泛的应用价值。     

环介导等温扩增技术检测小肠结肠炎耶尔森氏菌的研究

采用环介导等温扩增技术(Loop-mediated isothermal amplification,LAMP)快速检测小肠结肠炎耶尔森氏菌(Yersinia enterocolitica)。以小肠结肠炎耶尔森氏菌(AY004311.1)Ail基因序列作为靶序列,设计内、外引物,通过肉眼观察沉淀判断检测结果。结果表明:LAMP检测小肠结肠炎耶尔森氏菌的灵敏度为6.3cfu/mL,人工污染鸡肉的检出限为340cfu/g。PCR检测小肠结肠炎耶尔森氏菌的灵敏度为630cfu/mL,人工污染鸡肉的检出限为3.4×104cfu/g。采用试剂盒法提取DNA,从样品处理到报告结果,LAMP方法耗时2h,PCR方法耗时3h。因此,LAMP检测小肠结肠炎耶尔森氏菌的灵敏度高,耗时短,特异性好,操作简便,无需特殊的仪器设备,适合在我国广大基层实验室开展应用,为快速检测食源性致病菌构建了一个新的技术平台。     

Development of a loop-mediated isothermal amplification assay for detecting Listeria monocytogenes prfA in milk

A rapid and sensitive loop-mediated isothermal amplification (LAMP) assay was developed for detecting Listeria monocytogenes prfA in milk. The inclusivity of 23 L. monocytogenes and the exclusivity of 16 non-L. monocytogenes strains were both 100% in the assay. The limit of detection (LoD) of the LAMP assay in Listeria enrichment broth (LEB) was 2.22 CFU/mL after 12 h and 24 h of incubation. The LoDs of the LAMP assay in LEB with artificially contaminated milk (LEB-M) incubated for 12 h (2.22×10¹ CFU/mL) and 24 h (2.22 CFU/mL) were lower than those of the PCR and real-time PCR assays. Comparison of the LoDs in LEB with those in LEB-M showed that the LAMP assay was less influenced by the milk compounds than the real-time PCR assay. Our results indicate that the LAMP assay can be utilized as a potential screening tool for L. monocytogenes in milk.     

小麦成分环介导等温扩增现场快速检测方法的建立和应用

目的建立环介导等温扩增(loop-mediated isothermal amplification,LAMP)方法现场快速检测小麦过敏原成分的分析方法,有助于食品安全突发事件现场和食品企业生产过程中的样品检测小麦成分。方法运用LAMP建立食品过敏原小麦成分LAMP现场快速检测方法,采用实时浊度仪和显色法进行结果判断,并进行反应条件优化。结果本方法特异性强,对35个对照样品无交叉反应;方法的灵敏度高,检测限可达到0.01%小麦;方法稳定性好,对0.1%和0.01%小麦样品重复检测结果稳定。通过对市场采集70份样品检测结果显示,该方法与食品标签标示的过敏原成分结果一致。结论本方法操作简单,检测时限短,结果判断准确,可用于过敏原小麦成分的快速检测。     

Rapid and sensitive detection of Pseudomonas aeruginosa in bottled water by loop-mediated isothermal amplification

A loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Pseudomonas aeruginosa in bottled water was developed and evaluated. Four primers, including two outer primers and two inner primers, were specially designed by targeting the ecfX gene. The LAMP assay showed high specificity, with no false-positive or false-negative results among the 108 bacterial strains tested, including 81 strains of P. aeruginosa. The detection limit in pure culture was 15.7 CFU/mL, which is approximately 100-fold more sensitive than that of ecfX-PCR. In artificially contaminated water samples spiked with low level (3.1 CFU/250 mL) of P. aeruginosa, the LAMP assay could detect the target organisms accurately after 10 h of enrichment, in contrast to 12 h of enrichment for ecfX-PCR. In the case of bottled water samples, 9.17 % (10/109) of the samples were found to be positive by LAMP, which was in accordance with the results from GB 8538-2008 method. The LAMP assay established in this study is a simple, sensitive, and rapid protocol for the detection of P. aeruginosa. It provides an important diagnostic tool for the drinking water produce industry and regulatory agencies to better control potential risks associated with consumption of bottled water.     

Development and application of loop-mediated isothermal amplification assays on rapid detection of various types of staphylococci strains

A loop-mediated isothermal amplification (LAMP) method for rapid detection of various Staphylococcus strains and associated antibiotic resistance determinant had been developed and evaluated in this study. Six primers, including outer primers, inner primers and loop primers, were specially designed for recognizing eight distinct sequences on three targets: 16SrRNA, femA and mecA.. Forty-one reference strains, including various species of gram-negative and -positive isolates, were included in this study to evaluate and optimize LAMP assays. The optimal reaction condition was found to be 65 °C for 45 min, with detection limits at 100 fg DNA/tube and 10 CFU/reaction for 16S rRNA, 100 fg DNA/tube and 10 CFU/reaction for femA, 1 pg DNA/tube and 100 CFU/reaction for mecA, respectively. Application of LAMP assays were performed on 118 various types of Staphylococcus isolates, the detection rate of LAMP assays for the 16SrRNA, femA and mecA was 100% (118/118), 98.5% (64/65) and 94.3% (66/70), and the negative predictive value (NPV) was 100%, 98.1% and 92.3% respectively; with a 100% positive predictive value (PPV) for all three targets. In conclusion, LAMP assays were demonstrated to be useful and powerful tools for rapid detection of various Staphylococcus strains, and undoubtedly, the rapidness, technical simplicity, and cost-effectiveness of LAMP assays will demonstrate broad application for bacteriological detection of food-borne Methicillin-resistant Staphylococcus (MRS) isolates.     

环介导等温扩增技术在病原微生物快速检测中的研究进展

病原微生物是危害人类健康的最主要影响因素之一, 快速、准确地鉴定病原微生物对保障人类健康具有重要意义。环介导等温扩增技术(loop-meditated isothermal amplification, LAMP)是一种新型的等温核酸扩增技术, 具有特异性高、灵敏度高、快速、经济、简便的优点。可以与其他新技术结合, 弥补其引物设计困难和易被污染的不足, 使其能够在更多领域应用, 尤其是在资源贫乏地区的基因检测以及食品的快速检测和基层检测。本文就环介导等温扩增的基本原理、特点及在病原微生物检测中的应用对近些年的研究进展进行综述, 并对环介导等温扩增未来发展方向进行展望, 为环介导等温扩增技术的进一步发展提供参考。     

Development and evaluation of a loop-mediated isothermal amplification assay for the rapid detection of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> in food

A loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Staphylococcus aureus in food was developed and evaluated. Heat-stable nuclease (nuc) gene was the target sequence amplified by LAMP. LAMP includes primer design, amplification at 64 °C, macroscopic observation, and estimation of the results. The LAMP detection of S. aureus was sensitive up to 1.25 CFU per reaction tube, with a detection limit of 10.3 CFU per reaction tube in the artificial contamination test, and the experiment took less than 1 h. Sensitivity of the method in Staphylococcus aureus detection was 100%, with 97.93% specificity and a 98.5% match ratio when compared using Chinese National Standard. The LAMP method is superior to the Chinese National Standard method in terms of sensitivity, cost, testing time, and convenience.     

环介导等温扩增技术快速检测肉及肉制品中的牛源性成分

本文以牛源性线粒体细胞色素B(cyt B)基因为模板,采用软件Primer Explorer Version 4设计并筛选出LAMP特异性扩增引物,通过反应体系优化建立了肉及肉制品中牛源性成分的环介导等温扩增技术检测方法,对该方法的灵敏度、特异性以及稳定性进行了验证,同时采用国标方法和商品化检测试剂盒对市售的12种牛肉及牛肉制品进行对比检测。结果表明该检测方法具有高灵敏度、高特异性和高稳定性,适用于实际的生鲜肉制品及加工肉制品中牛源性成分的鉴定。