副溶血弧菌LAMP检测方法的建立

副溶血弧菌(Vibrio parahaemolyticus)是一种能引起食源性疾病的重要病原菌。首次将一种新颖的核酸扩增技术-环介导等温扩增技术（Loop-Mediated Isothermal Amplification, LAMP）应用于副溶血弧菌的快速检测。针对副溶血弧菌不耐热溶血毒素基因（tlh）设计四条特异性引物（两条内引物和两条外引物）进行LAMP扩增,对扩增反应进行优化,最佳反应时间为60 min,反应温度为60 ℃。对12种细菌共28株菌进行LAMP扩增,仅14株副溶血弧菌得到阳性扩增结果,证明引物具有很高的特异性。副溶血弧菌基因组DNA和纯培养物的检测灵敏度分别约为90 fg和24 cfu/mL。对模拟食品样品进行直接检测,检测限为89 cfu/g。结果表明,该方法检测副溶血弧菌特异性强、灵敏度高,并且操作简便、检测成本低,1 h即可完成,有望发展成为快速检测副溶血弧菌的有效手段。     

环介导等温扩增联合横向流动试纸条可视化检测志贺氏菌

【目的】将环介导等温扩增技术(LAMP)与横向流动试纸条(LFD)联合应用,建立一种可应用于志贺氏菌快速检测的LAMP-LFD技术。【方法】以福氏志贺氏菌的侵袭性质粒抗原H(ipa H)基因为检测靶标设计3对特异性引物(其中上游内引物Sfl-ipa H-FIP由生物素标记),进行LAMP反应;同时设计1条异硫氰酸荧光素(FITC)标记的探针Sfl-ipa H-HP,与获得的LAMP产物进行特异性杂交,杂交产物经LFD完成检测。【结果】优化后的LAMP反应条件为63°C 40 min,加上LFD结果判读共需50 min。LAMP-LFD方法能够特异性检测出福氏志贺氏菌,而对肠炎沙门氏菌等其它4种导致腹泻的致病菌和创伤弧菌等5种常见食物源性致病菌,以及4株不同大肠杆菌的检测结果呈阴性。该方法针对福氏志贺氏菌的检测灵敏度为1.0×10~2 CFU/m L或4 CFU/反应,针对人工污染鲤鱼肠组织的检测灵敏度是5.0×10~2 CFU/m L,是以LAMP外引物Sfl-ipa H-F3/Sfl-ipa H-B3的常规PCR方法的100倍。【结论】建立的LAMP-LFD技术具有操作简单、检测快速准确、检测成本低等优点,有望在志贺氏菌的常规监测和即时检测中被普及使用。     

Species and sex identification of Formosa landlocked salmon using loop-mediated isothermal amplification

Species and sex identification are among the most important parameters for conservation management. However, it is extremely difficult to perform such identification in Formosa landlocked salmon (Oncorhynchus masou formosanus). Both sexual dimorphism in landlocked dwarf form Formosa landlocked salmon and morphological difference among cherry salmon complex are minimal. We developed a simple, rapid and noninvasive method for identifying sex and species of this critically endangered species using a loop-mediated isothermal amplification (LAMP) technique. The LAMP assay showed the advantage of simple detection (evaluated by visual inspection), rapid reaction time (< 1 h), isothermal condition (less equipment required) and high efficiency (only 0.5-5 pg of DNA was required in the reaction mixture). Therefore, the method is more economical and practical than PCR. The LAMP assay can be easily performed in the field and is a valuable tool for detecting sex ratios in wild populations and identifying species in commercial imports. This is the first application of LAMP in identifying species and sex of salmonids as far as we know and clearly shows the potential application of LAMP in molecular ecology and conservation efforts.     

Loop‐mediated isothermal amplification method for rapid detection of Vibrio alginolyticus,the causative agent of vibriosis in mariculture fish

Aims: The purpose of this study was to develop a loop‐mediated isothermal amplification (LAMP) method for the rapid, sensitive and simple detection of Vibrio alginolyticus in mariculture fish. Methods and Results: LAMP primers were designed by targeting the gyrB gene. With Bst DNA polymerase, the target DNA can be clearly amplified for 60 min at 64°C in a simple water bath. The detection sensitivity of the LAMP assay for the detection of V. alginolyticus is about 3·7 × 10² CFU ml^?1 (3·7 CFU per reaction). LAMP products could be judged with agar gel or naked eye after the addition of SYBR Green I. There were no cross‐reactions with other bacterial strains indicating a high specificity of the LAMP. The LAMP method was applied to detect V. alginolyticus‐infected fish tissues effectively. Conclusions: The LAMP established in this study is a simple, sensitive, specific, inexpensive and rapid protocol for the detection of V. alginolyticus. Significance and Impact of the Study: This LAMP method provides an important diagnostic tool for the detection of V. alginolyticus infection both in the laboratory and field.     

DNA环介导恒温扩增技术快速检测霍乱弧菌

霍乱弧菌是一种重要的食源性致病菌,主要引起急性肠道传染病,其快速检测具有重要意义。根据霍乱弧菌的mdh管家基因序列,设计2对特异性检测引物,利用DNA环介导恒温扩增技术(Loop-mediated isothermal amplification,LAMP),经反应体系优化,成功建立了霍乱弧菌的LAMP快速检测方法。该方法最佳反应温度为65℃,60min完成检测,对培养菌的检测限为25CFU/mL,污染食品中霍乱弧菌的检测限为32CFU/g。对33株同种或近源细菌进行LAMP检测,仅霍乱弧菌得到阳性扩增。LAMP方法实践应用结果表明,对1057份虾、蟹、牡蛎、肉类、人腹泻物等样本进行检测,共检出85份阳性,与国际标准(ISO TS21872-1-2007)检测结果的符合率为100%。结果表明,本研究建立的霍乱弧菌LAMP检测方法特异性强、灵敏度高、操作简便,有利于霍乱弧菌疫情的监测。     

Rapid and sensitive detection of Vibrio cholerae by loop-mediated isothermal amplification targeted to the gene of outer membrane protein ompW

Aims:  The present study was aimed to develop a loop-mediated isothermal amplification (LAMP) assay for rapid and specific detection of Vibrio cholerae .
Methods and Results:  A set of five designed primers that recognized specifically the V. cholerae ompW gene was used. The optimized time and temperature conditions for the LAMP assay were 75 min at 65°C, respectively. The LAMP method accurately identified 16 isolates of V. cholerae but did not detect 28 non- cholerae Vibrio isolates and 37 non- Vibrio bacterial isolates. The sensitivity of LAMP for V. cholerae detection in pure cultures was 2·2 × 10³ CFU ml⁻¹ or equivalent to 8 CFU per reaction. In the case of spiked shrimp samples without enrichment, the detection limit for V. cholerae was 2·2 × 10⁴ CFU g⁻¹ or equivalent to 20 CFU per reaction, while that of PCR was 100 CFU per reaction.
Conclusion:  The developed LAMP assay targeting ompW gene was rapid, specific and sensitive for V. cholerae detection.
Significant and Impact of the study:  The developed LAMP assay appears to be precise, accurate and a valuable tool for detection of V. cholerae . This assay can replace laborious biochemical tests for the identification of V. cholerae in contaminated food sample.     

Comparison of loop-mediated isothermal amplification and real-time PCR for the diagnosis of tuberculous pleurisy

Aims: Tuberculous pleurisy is an important cause of pleural effusions in areas with a high incidence of tuberculosis. In this study, we developed an IS1081‐based LAMP for the detection of Mycobacterium tuberculosis complex and investigated its usefulness in the diagnosis of tuberculous pleurisy. Methods and Results: Investigation of pleural effusion samples from patients with tuberculous pleurisy, majority of them smear‐/culture‐negative, and control individuals with non‐TB diseases showed that the LAMP assay with incubation time of 60 min has much higher specificity and the LAMP assay with incubation time of 90 min has significantly higher sensitivity in the diagnosis of tuberculous pleurisy, as compared with fluorescent real‐time PCR. Conclusions: The MTBC–LAMP is a useful assay for the diagnosis of tuberculous pleurisy, especially in pleural effusion smear‐/culture‐negative patients. Significance and Impact of the Study: Tuberculous pleural effusion usually contains low number of mycobacteria, which leads to low diagnostic sensitivity of acid‐fast staining and mycobacterial culture methods. In this study, we developed a simple and sensitive LAMP assay for the diagnosis of tuberculous pleurisy. This assay should have broad application in resource‐limited settings.     

Simple and specific detection of Bordetella holmesii by using a loop-mediated isothermal amplification assay

A loop-mediated isothermal amplification (LAMP) assay for simple detection of Bordetella holmesii was developed. This assay discriminates between B. holmesii and other Bordetella species and successfully detect B. holmesii DNA in nasopharyngeal swab samples from subjects with suspected pertussis. The LAMP assay results were in complete agreement with the results of previously published real-time PCR assay, indicating that the former is a powerful tool for the accurate diagnosis and surveillance of B. holmesii.     

The development of loop-mediated isothermal amplification combined with lateral flow dipstick for detection of Karlodinium veneficum

The aim of this study was to develop a loop-mediated isothermal amplification (LAMP) combined with a chromatographic lateral flow dipstick (LFD) assay to rapidly and specifically detect the Karlodinium veneficum ITS gene. Four groups of LAMP primers were specially designed to target the K. veneficum ITS gene. The LAMP-LFD detection limit was 7.4 pg/μL (approximately 6.5 cells/mL) of K. veneficum genomic DNA and was 10 times more sensitive than standard PCR. The LAMP-LFD method exhibited high specificity and accurately identified K. veneficum algal isolates, but not other algal isolates. To test the assay’s accuracy, samples from positive results were further analyzed by sequencing and phylogenetic analysis, all of which were identified as K. veneficum. Over all, the LAMP-LFD assay established in this paper can be used as a reliable and simple method to detect the K. veneficum.     

Development and evaluation of a loop‐mediated isothermal amplification assay for rapid detection of chicken anaemia virus

Aim: Chicken anaemia virus (CAV) causes an economically important viral disease in chickens worldwide. The main aim of this study was to establish a rapid, sensitive and specific loop‐mediated isothermal amplification (LAMP) assay for detecting CAV infection. Methods and Results: A set of four specific LAMP primers were designed based on the nucleotide sequence of the CAV VP2 gene, which encodes a nonstructural protein. These were used for the amplification of a specific target region of the VP2 gene. LAMP amplicons were successfully amplified and detected by DNA electrophoresis and by direct naked eye SYBR Green I visualization. A sensitivity test systematically demonstrated that the LAMP assay was superior to a conventional PCR assay with a minimum concentration limit of 100 fg compared to 10 ng for the conventional PCR. The specificity of the LAMP assay for CAV detection is consistent with conventional PCR. Using this established LAMP assay, infected and uninfected clinical samples obtained from an experimental farm were fully verified. Conclusions: A novel nucleic acid‐based approach of LAMP assay was successfully developed for detecting CAV infection. Significance and Impact of the Study: In this study, these results indicate that the developed LAMP assay herein for CAV detection is a time‐effective, simple, sensitive and specific test that can be used as an alternative approach in the future for large‐scaled diagnosis on the farm of CAV infection.     

Rapid, sensitive and simple detection method for koi herpesvirus using loop-mediated isothermal amplification

New methods were developed for the detection of koi herpesvirus (KHV, CyHV-3) by LAMP, which were compared with the PCR for specificity and sensitivity. We designed two primer sets targeting a specific sequence within the 9/5 PCR amplicon (9/5 LAMP) and the upper region of the Sph I-5 PCR amplicon ( Sph I-5 LAMP), including a sequence highly conserved among the strains. The amplification was monitored in real-time based on the increase in turbidity, with magnesium pyrophosphate as the by-product. The reactions were carried out under isothermal conditions at 65°C for 60 min. The detection limit of both LAMP was six copies, equal to the modified Sph I-5 PCR. No cross-reactivity with other fish pathogenic viruses and bacteria was observed. Sph I-5 LAMP was found to have a quicker response in terms of the reaction velocity than 9/5 LAMP. Therefore, we consider Sph I-5 LAMP to be superior for routine use. Additionally, LAMP was found applicable to crude extract from gills and other organs. LAMP methods are superior in terms of sensitivity, specificity, rapidity and simplicity, and are potentially a valuable diagnostic tool for KHV infections.     

Detection of phytoplasma by loop-mediated isothermal amplification of DNA (LAMP)

Napier stunt phytoplasma (16SrXI and 16SrIII) in eastern Africa is a serious threat to the expansion of Napier grass (Pennisetum purpureum) farming in the region, where it is widely cultivated as fodder in zero grazing livestock systems. The grass has high potential for bio-fuel production, and has been adopted by farmers as a countermeasure to cereal stem borer Lepidoptera, since it attracts and traps the insect. Diagnosis of stunt phytoplasma have been largely by nested polymerase chain reaction (nPCR) targeting the 16S rRNA gene. However, the method is laborious, costly and technically demanding. This investigation has developed a simpler but effective phytoplasma diagnostic tool, called; loop-mediated isothermal amplification of DNA (LAMP). The assay was tested on 8 symptomatic and 8 asymptomatic plants, while its detection limit was compared to nested PCR using samples serially diluted from 3 ng/μl to 0.38 pg/μl. Molecular typing of LAMP products was determined by BsrI restriction digestion and Southern blot analysis. The assay sensitivity, positive and negative predictive values were estimated, while the specificity was tested on 11 phytoplasma groups. LAMP was specific to 5 phytoplasma groups: 16SrVI, X, XI and XVI. BsrI restriction digestion produced two predicted fragments, and there was specific binding of probe DNA to the LAMP amplicons in Southern blot analysis. The assay sensitivity was 100%, while the positive and negative predictive values were 63 and 100% respectively. LAMP was 20-fold more sensitive than nested PCR. This study validates LAMP for routine diagnosis of Napier stunt and other closely related phytoplasmas.     

环介导等温扩增技术快速检测施罗氏弧菌(Vibrio shilonii)

【背景】近年来,珊瑚白化事件频有发生,面临着严重衰退。由气候变化引起的珊瑚病原菌快速增殖是导致珊瑚白化的主要因素之一。施罗氏弧菌是枇杷珊瑚的致病菌,能侵入珊瑚虫体内而使珊瑚白化死亡。【目的】优化并建立一种钙黄绿素显色法快速检测珊瑚致病菌施罗氏弧菌的环介导等温扩增(Loop-mediatedisothermalamplificaiton,LAMP)检测技术。【方法】以枇杷珊瑚致病菌施罗氏弧菌为研究对象,针对施罗氏弧菌的rpoD (RNA polymerase subunit D)基因设计6条特异性扩增引物,建立LAMP检测体系并检测其特异性和灵敏度,同时对LAMP法、常规PCR和荧光定量PCR3种检测方法进行比较分析。【结果】供检测的10个样品菌株中,施罗氏弧菌反应结果为阳性,呈亮绿色,其他9株包括阴性对照(灭菌水为模板)反应结果为阴性,呈浅橙黄色;同时,所建立的钙黄绿素-LAMP方法最低检测限度为3.641×10~3 cps/mL,具有与荧光定量PCR等同的灵敏度和准确性,是常规PCR最低检测限度的0.1%;此外,通过模拟野外海水样品检测发现,钙黄绿素-LAMP方法对海水样品中施罗氏弧菌的检测限度可达1.3×10~2 CFU/mL。【结论】建立的钙黄绿素-LAMP检测技术具有很好的特异性、灵敏度和准确性,其操作方法简单、方便,无需昂贵仪器,适用于野外现场珊瑚致病菌施罗氏弧菌的快速检测。     

Tolerance of loop-mediated isothermal amplification to a culture medium and biological substances

To simplify the molecular detection of micro-organisms, we evaluated the tolerance of loop-mediated isothermal amplification (LAMP) to a culture medium and some biological substances. The sensitivity of LAMP was less affected by the various components of the clinical samples than was polymerase chain reaction (PCR); therefore, DNA purification from samples could be omitted.     

Development and evaluation of loop-mediated isothermal amplification assay for rapid and sensitive detection of canine parvovirus DNA directly in faecal specimens

Aims: To develop a specific and highly sensitive loop-mediated isothermal amplification (LAMP) technique for the rapid detection of canine parvovirus (CPV) DNA directly in suspected faecal samples of dogs by employing a simple method of template preparation. Methods and Results: LAMP reaction was developed by designing two sets of outer and inner primers, which target a total of six distinct regions on VP2 gene of CPV. The template DNA was prepared by a simple boiling and chilling method. Of the 140 faecal samples screened by the developed LAMP and the conventional PCR assays, 104 samples (74·28%) were found positive by LAMP, whereas 81 samples (57·85%) were found positive by PCR. The specificity of the LAMP assay was tested by cross-examination of common pathogens of dogs and further confirmed by sequencing. The detection limit of the LAMP was 0·0001 TCID(50) ml(-1) , whereas the detection limit of the PCR was 1000 TCID(50) ml(-1) . Conclusions: The developed LAMP assay detects CPV DNA in faecal specimens directly within an hour by following a simple and rapid boiling and chilling method of template preparation. The result also shows that the developed LAMP assay is specific and highly sensitive in detecting CPV. Significance and Impact of the Study: The result indicates the potential usefulness of LAMP which is a simple, rapid, specific, highly sensitive and cost-effective field-based method for direct detection of CPV from the suspected faecal samples of dogs.     

Development of a loop-mediated isothermal amplification assay for porcine circovirus type 2

In this study,the loop-mediated isothermal amplification(LAMP)method was used to develop a rapid and simple detection system for porcine circovirus type 2(PCV2).According to the PCV2 sequences published in GenBank,multiple LAMP primers were designed targeting conserved sequences of PCV2.Using the DNA extracted from PCV2 isolates HUN-09 and SD-09 as the template,LAMP reactions in a PCV2 LAMP system was performed,the amplification products were detected by adding SYBR Green I and could be observed directly by the naked eye.The results showed highly-efficient and specific amplification in 30 min at 63℃ with a LAMP real-time turbidimeter.Furthermore,PCV2 DNA templates,with a detection limit of 5.5×10-5ng of nucleic acid,indicated that this assay was highly sensitive.The results obtained with the naked eye after SYBR Green I staining were consistent with those detected by the real-time turbidimeter,showing the potential simplicity of interpretation of the assay results.The LAMP assay appeared to have greater accuracy than PCR and virus isolation for the analysis of 18 clinical samples.In addition it offers higher specificity and sensitivity,shorter reaction times and simpler procedures than the currently available methods of PCV2 detection.It is therefore a promising tool for the effective and efficient detection of PCV2.     

四重PCR检测副溶血性弧菌及其毒力基因方法的建立

【目的】建立同时检测副溶血性弧菌tox R、tdh、trh、tlh基因的四重PCR快速检测方法。【方法】分别以副溶血性弧菌的tox R、tdh、trh、tlh 4个基因为靶基因,设计4对特异性引物,对4对引物浓度和退火温度进行优化,获得最佳引物比例和扩增条件,建立快速检测致病性副溶血性弧菌的四重PCR体系。通过特异性验证、灵敏度验证以及模拟样品检测进行方法确认。【结果】四重PCR体系扩增条带与预期相符,即115 bp(tox R)、244 bp(tdh)、418 bp(trh)、759 bp(tlh)4个目的条带;用74株副溶血性弧菌和37株非目标菌的测试结果表明,所建立的方法有良好的特异性。该方法对模板DNA的检测灵敏度为50μg/L,纯培养物的检测灵敏度为6.7×103 CFU/m L;副溶血性弧菌含量为1.36 CFU/g的人工模拟样品增菌6 h后,tox R、tlh、tdh、trh 4个基因可同时被检出。【结论】该方法可实现同时检测携带tox R、tdh、trh、tlh 4种基因的副溶血性弧菌,对开展致病性副溶血性弧菌的检测研究具有一定现实意义。     

The development of rapid real-time PCR detection system for Vibrio parahaemolyticus in raw oyster

Aims:  To develop a new rapid real-time polymerase chain reaction (PCR) based detection system for Vibrio parahaemolyticus ( V. parahaemolyticus ) applicable to raw oyster samples.
Methods and Results:  V. parahaemolyticus cells were artificially inoculated to oysters. Samples were homogenized in 100 ml of sterile saline water and serially diluted to 1·5 CFU ml⁻¹ level. One millilitre of diluents was centrifuged and the pellet was resuspended with 100  μ l of de-ionized water. DNA was extracted by boiling for 20 min, and 0·5  μ l was used as a template for PCR reaction. Real-time PCR was performed with TMC-1000 system (1  μ l PCR system). The detection system was found to achieve detection limit of 1·5 CFU g⁻¹ for V. parahaemolyticus . Furthermore, the specificities of these assay systems were confirmed with more than 20 bacterial strains, including various Vibrio species.
Conclusions:  Rapid and sensitive food-borne pathogen detection techniques for V. parahaemolyticus is important to the food industry and consumers. The direct detection of V. parahaemolyticus from food is possible with micro real-time PCR system.
Significance and Impact of the Study:  This study shows that oyster samples can be tested for V. parahaemolyticus with a rapid, specific and simple procedure.