水霉菌环介导等温扩增检测方法的建立

【目的】建立一种快速简单检测水霉病病原菌的方法。【方法】针对水霉菌ITS区基因序列设计4条特异性引物,包括两条外引物和两条内引物,优化反应条件,观察检测结果。对该方法的特异性和敏感性进行研究。【结果】建立了环介导等温扩增技术检测水霉菌的方法,确定了其最适反应条件。该方法能够检测到浓度低至103个/mL的水霉菌孢子,其灵敏度是普通PCR方法的100倍。【结论】建立的检测水霉菌的LAMP技术,具有操作简便快速等特点,可用做特异性水霉及其孢子的快速鉴定。     

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.     

Rapid and simple detection of Clostridium botulinum types A and B by loop-mediated isothermal amplification

Aims:  To develop a convenient and rapid detection method for toxigenic Clostridium botulinum types A and B using a loop-mediated isothermal amplification (LAMP) method.
Methods and results:  The LAMP primer sets for the type A or B botulinum neurotoxin gene, BoNT / A or BoNT / B , were designed. To determine the specificity of the LAMP assay, a total of 14 C. botulinum strains and 17 other Clostridium strains were tested. The assays for the BoNT/A or BoNT/B gene detected only type A or B C. botulinum strains, respectively, but not other types of C. botulinum or strains of other Clostridium species. Using purified chromosomal DNA, the sensitivity of LAMP for the BoNT/A or BoNT/B gene was 1 pg or 10 pg of DNA per assay, respectively. The assay times needed to detect 1 ng of DNA were only 23 and 22 min for types A and B, respectively. In food samples, the detection limit per reaction was one cell for type A and 10 cells for type B.
Conclusions:  The LAMP is a sensitive, specific and rapid detection method for C. botulinum types A and B.
Significance and Impact of the Study:  The LAMP assay would be useful for detection of C. botulinum in environmental samples.     

应用等温环介导扩增方法检测蜜蜂残翅病毒的研究

本文的目的在于建立用于临床检测残翅病病毒(Deformed wing virus,DWV)的等温环介导扩增技术(Loopmediated isothermal amplification,LAMP),为该疾病的预防和控制提供理论依据。在DWV基因保守序列设计4条引物,探究LAMP扩增的最优条件,并与常规的PCR(polymerase chain reaction)检测方法进行比较。建立的LAMP方法检测下限为0.89 pg,灵敏度比PCR高100倍而且特异性好。临床检测显示建立的LAMP方法可行、准确、方便、灵敏。针对DWV的LAMP建立的检测方法为养蜂生产第一线检测和预防DWV提供了技术支持,有一定的应用价值。     

环介导等温扩增技术用于检测幽门螺旋杆菌CagA基因

目的 建立环介导等温扩增技术（LAMP）用于检测唾液和胃液中幽门螺旋杆菌CagA基因。方法 收集临床阳性样本并用荧光定量PCR检测,然后根据幽门螺旋杆菌的CagA基因序列设计LAMP引物并优化反应条件。通过LAMP检测来验证引物的特异性,并通过检测稀释的模板来验证引物的灵敏性。同时加入钙黄绿素使产物显色,使CagA基因检测可视化。最后,用LAMP方法检测收集的临床样本,并计算阳性率,应用配对卡方检验比较LAMP和荧光定量PCR的统计学差异。结果 LAMP体系的特异性和灵敏性均较好,对CagA基因检测的灵敏性是102 IU/mL。另外,利用钙黄绿素进行产物检测的效果和电泳结果相当。通过对临床样本的检测,CagA阳性样本的LAMP检测结果和荧光定量PCR差异有统计学意义（P＜0.05）。结论 LAMP对CagA基因的检测具有较好的特异性和灵敏性,操作简便,在基层医院有较好的应用前景。     

Loop-mediated isothermal amplification targeting the apxIVA gene for detection of Actinobacillus pleuropneumoniae

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method performed under isothermal conditions with high specificity and efficiency. We developed a diagnostic method based on LAMP for detection of Actinobacillus pleuropneumoniae . Using six specific primers targeting the apxIVA gene, the LAMP assay rapidly amplified the target gene within 30 min, requiring only a laboratory water bath for the reaction to occur. The resulting amplificon was visualized by adding SYBR Green I to the mixture. The results obtained from testing 15 A. pleuropneumoniae reference strains and other seven bacterial species strains showed that the LAMP was as specific as and 10 times more sensitive than nested PCR. Sixty-five tonsil samples were collected from 65 healthy pigs. All the samples were negative for A. pleuropneumoniae by immunomagnetic separation-based (IMS) bacterial isolation, nested PCR and LAMP, respectively. Meanwhile, 115 tonsil samples were also collected from 115 pigs with apparent respiratory problems. Twenty-two were positive by IMS bacterial isolation. All the samples that were positive by IMS bacterial isolation were also positive by nested PCR and LAMP. The LAMP assay demonstrated exceptionally higher sensitivity than nested PCR by picking up 14 additional positive cases (χ² test, P <0.0001); we concluded that LAMP was a highly sensitive and reliable method for detection of A. pleuropneumoniae infection.     

NLF2 gene expression in the endometrium of patients with implantation failure after IVF treatment

We developed and evaluated the specificity and sensitivity of a loop-mediated isothermal amplification (LAMP) method for rapid detection of the multidrug-resistance gene cfr. A pair of outer primers and a pair of inner primers and one loop primer were specially designed for recognizing seven distinct sequences on the target cfr gene. The amplification reaction was performed within only 35 min under isothermal conditions at 63 °C in a regular water bath with visual measurement. The LAMP assay showed higher sensitivity than the conventional PCR, with a detection limit of 1 pg per tube of chicken Staphylococcus sciuri genomic DNA. The detection rate of cfr gene for 50 Staphylococcus clinical strains by LAMP assays was 16% and appeared 100% consistence with the result by PCR method. The LAMP method reported here demonstrated a potential and valuable means for detection of the multidrug-resistance gene cfr: easy, rapid, visual, specific, accurate and sensitive, especially useful for on-the-spot investigation.     

环介导等温可视扩增检测牛分枝杆菌方法的建立

目的:建立一种快速简便检测牛分枝杆菌的方法。方法:根据已发表的牛分枝杆菌特殊基因序列,设计并合成6对特异扩增牛分枝杆菌特异性基因片段的引物,通过条件优化,建立针对牛分枝杆菌的环介导等温扩增(LAMP)检测法,测定其特异性和敏感性,并对采集的牛临床样品的DNA分别进行检测。结果:采用该法只检出牛分枝杆菌,检测的最低拷贝数为1×102拷贝/μL。结论:建立的LAMP方法简便、快速、特异性高,可用于临床上牛分枝杆菌的快速检测。     

Establishment of loop-mediated isothermal amplification (LAMP) for rapid detection of Brucella spp. and application to milk and blood samples

Brucella spp. are facultative intracellular bacteria that infect humans and animals. In this study, the loop-mediated isothermal amplification (LAMP) was used to detect the Brucella-specific gene omp25. Reaction conditions were optimized as temperature 65°C, reaction time 60 min, Mg(2+) concentration 8.0 mmol/L, polymerase content Bst DNA, 0.5 μL, deoxyribonucleotide concentration 1.6 mmol/L, and inner/outer primer ratio 1:8. The LAMP method was evaluated with 4 Brucella species and 29 non-Brucella bacteria species. Positive reactions were observed on all the 4 Brucella species but not on any non-Brucella species. The limit of detection of the LAMP method was 3.81 CFU Brucella spp. Using the LAMP method, 7 of 110 raw milk samples and 5 of 59 sheep blood samples were detected positive of Brucella spp. Results indicated that LAMP is a fast, specific, sensitive, inexpensive, and suitable method for diagnosis of Brucella spp. infection.     

Sensitive and rapid detection of Flavobacterium columnare in channel catfish Ictalurus punctatus by a loop-mediated isothermal amplification method

AIMS: To evaluate the loop-mediated isothermal amplification method (LAMP) for rapid detection of Flavobacterium columnare and determine the suitability of LAMP for rapid diagnosis of columnaris infection in channel catfish, Ictalurus punctatus. METHODS AND RESULTS: A set of four primers, two outer and two inner, were designed specifically to recognize 16S ribosomal RNA gene of this pathogen. Bacterial genomic DNA templates were prepared by hot lysis in a lysis buffer. Amplification of the specific gene segments was carried out at 65 degrees C for 1 h. The amplified gene products were analysed by agarose gel electrophoresis and detected by staining gels with ethidium bromide. A PCR assay was also included in this study. Our results demonstrate that the ladder-like pattern of bands from 204 bp specific to the Fl. columnare 16S ribosomal RNA gene was amplified. The detection limit of the LAMP assay was comparable to that of PCR in prepared genomic DNA reactions. In addition, this optimized LAMP assay was able to detect the Fl. columnare 16S ribosomal RNA gene in experimentally infected channel catfish. CONCLUSIONS: The LAMP assay for Fl. columnare detection in channel catfish was established. SIGNIFICANCE AND IMPACT OF THE STUDY: Because LAMP assay is a rapid, sensitive, specific, simple and cost-effective assay for Fl. columnare detection in channel catfish, it is useful for rapid diagnosis of Fl. columnare in fish hatcheries and the field.     

Principle of LAMP method--a simple and rapid gene amplification method

So far nucleic acid test (NAT) has been employed in various fields, including infectious disease diagnoses. However, due to its complicated procedures and relatively high cost, it has not been widely utilized in many actual diagnostic applications. We have therefore developed a simple and rapid gene amplification technology, Loop-mediated Isothermal Amplification (LAMP) method, which has shown prominent results of surpassing the performance of the conventional gene amplification methods. LAMP method acquires three main features: (1) all reaction can be carried out under isothermal conditions; (2) the amplification efficiency is extremely high and tremendous amount of amplification products can be obtained; and (3) the reaction is highly specific. Furthermore, developed from the standard LAMP method, a rapid LAMP method, by adding in the loop primers, can reduce the amplification time from the previous 1 hour to less than 30 minutes. Enormous amount of white precipitate of magnesium pyrophosphate is produced as a by-product of the amplification, therefore, direct visual detection is possible without using any reaction indicators and detection equipments. We believe LAMP technology, with the integration of these features, can rightly apply to clinical genetic testing, food and environmental analysis, as well as NAT in different fields.     

水产品中创伤弧菌DNA环介导恒温扩增快速检测方法的建立及初步应用

创伤弧菌是一种重要的食源性致病菌,主要存在于河口和海洋环境中,严重危害水产养殖业的发展和人类健康。建立快速、准确、易操作的检测方法对防控创伤弧菌的传染,保障水产养殖业发展和增强食品安全意义重大。基于创伤弧菌vvHA基因,利用一种新型的核酸扩增技术-环介导恒温扩增(loop-mediated isothermal amplification,LAMP),建立了创伤弧菌LAMP快速检测方法。对11种共46株细菌进行扩增,仅创伤弧菌为LAMP阳性结果,说明LAMP方法具有高度特异性。灵敏度试验结果表明,对创伤弧菌纯培养菌的检测灵敏度为15CFU/ml,对污染食品中创伤弧菌的检测灵敏度为24CFU/g。此法40~60min内即可完成检测,检验检疫实践证明:LAMP方法操作简便、特异性强、灵敏度高且成本低廉,具有良好的应用前景。     

Development and evaluation of loop-mediated isothermal amplification for rapid detection of Haemophilus parasuis

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method performed under isothermal conditions and has a high specificity and efficiency. We developed a LAMP assay targeting the 16S rRNA gene for rapid detection of Haemophilus parasuis. The results obtained from testing 31 H. parasuis strains and 28 other bacterial species strains showed that LAMP was as specific as, and more sensitive than, nested PCR. Fifty-five lung samples were collected from 55 healthy pigs. All the samples were negative for H. parasuis by bacterial isolation, nested PCR and LAMP, respectively. In addition, 122 lung samples were collected from 122 pigs with apparent respiratory problems. Sixty-five were positive by bacterial isolation. All the samples that were positive by bacterial isolation were also positive by nested PCR and LAMP. The LAMP assay demonstrated higher sensitivity than nested PCR, picking up 16 additional cases. The LAMP assay also gave a same result compared with the nested PCR when the two assays were used, respectively, to detect H. parasuis from samples obtained from experimentally infected pigs. We concluded that LAMP is a highly sensitive and reliable method for detection of H. parasuis infection.     

苏云金芽孢杆菌Cry1A(b)抗虫基因LAMP检测方法的建立与应用

以转基因玉米MON810为模板,针对Cry1A(b)抗虫基因核酸保守序列设计特异性引物,建立LAMP检测体系。对该体系的可行性、灵敏性、特异性进行分析,并应用于转基因产品的检测。研究结果显示该方法快速简单、灵敏度特异性高、结果可视化,可应用于转基因产品中Cry1A(b)基因的初步筛选。     

鸭瘟病毒LAMP可视化检测方法的建立

目的:建立一种快速、简便、特异性高的鸭瘟病毒(DPV)环介导等温扩增(LAMP)检测方法。方法:根据Gen Bank中DPVUI6基因的保守序列设计一套特异性引物,并对反应条件进行优化,建立DPV的LAMP可视化检测方法。结果:建立的LAMP方法对其他鸭常见病原体无扩增反应;可通过肉眼观察颜色直接判定结果;敏感性可达0.1fg,是常规PCR方法的100倍;扩增反应只须在常规水浴锅中进行,可在1 h内完成。结论:建立的DPV LAMP方法简便、快速、灵敏、特异,可用于DPV感染的快速检测。     

Rapid and simple detection of eight major periodontal pathogens by the loop-mediated isothermal amplification method

Loop-mediated isothermal amplification (LAMP) was applied to develop a rapid and simple detection system for eight periodontal pathogens: Aggregatibacter (Actinobacillus) actinomycetemcomitans, Campylobacter rectus, Eikenella corrodens, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Treponema denticola and Tannerella forsythia. Primers were designed from the 16S ribosomal RNA gene for each pathogen, and the LAMP amplified the targets specifically and efficiently under isothermal condition at 64 degrees C. To simplify the manipulation of LAMP examination, boiled cells and intact cells suspended in phosphate-buffered saline (PBS) were tested as templates besides extracted DNA template. The detection limits were 1-10 cells per tube using extracted DNA template. However, LAMP methods using boiled cells and intact cells required 10-100 and 100-1000 cells per tube, respectively. LAMPs for A. actinomycetemcomitans, P. gingivalis and P. intermedia were then applied to clinical plaque samples, and the method demonstrated equal or higher sensitivity compared with the conventional real-time PCR method. These findings suggest the usefulness of the LAMP method for the rapid and simple microbiological diagnosis of periodontitis, and the possibility of LAMP examination without the DNA extraction step.     

快速检测中药材乌梢蛇LAMP方法的建立

乌梢蛇作为一种名贵中药材,市面上伪品较多,干燥熏黑处理后的样品,更是真伪难辨。本研究致力于开发一套基于环介导等温扩增(loop-mediated isothermal amplification,LAMP)为基础的快速筛查乌梢蛇的方法。本研究以乌梢蛇12srRNA基因序列为基础设计并筛选出1套LAMP引物。通过调整反应条件,建立了对乌梢蛇LAMP的检查方法。结果显示,62℃下连续反应15min左右出现典型的"S"型荧光吸收曲线,实现了对乌梢蛇12srRNA基因序列的特异扩增。根据LAMP灵敏度高的特点,本研究简化了DNA的提取方法,缩短了检测的时间。相对于常规的PCR方法,本研究建立的以快速DNA提取为基础的乌梢蛇LAMP快速筛查方法具有简单、快速、灵敏、对设备要求低等特点,适用于对中药材乌梢蛇的快速筛查。     

Real-time quantitative LAMP (loop-mediated isothermal amplification of DNA) as a simple method for monitoring ammonia-oxidizing bacteria

Loop-mediated isothermal amplification (LAMP) of DNA is a novel technique for the amplification of DNA under isothermal conditions. For the first time, we applied this method to develop a simple and quantitative monitoring method for environmental microorganisms targeting amoA gene in ammonia-oxidizing bacteria. Quantitative analysis was performed first by measuring fluorescence derived from an intercalation dye using a real-time thermal cycler, and then by measuring the turbidity of the reaction solution using a real-time turbidimeter. As a result, it was possible to quantify the initial amoA DNA concentration from an environment with a sensitivity down to 10(2) DNA copies of target DNA and a dynamic range of 7-9 orders in magnitude. Background DNA from nontargeted bacteria (Pseudomonas denitrificans) that does not encode amoA gene did not affect the quantitative capability of LAMP. Over results suggested that the real-time LAMP is effective for monitoring microorganisms and their gene expression in environments.     

Evaluation of a loop-mediated isothermal amplification method for rapid detection of channel catfish Ictalurus punctatus important bacterial pathogen Edwardsiella ictaluri

Channel catfish Ictalurus punctatus infected with Edwardsiella ictaluri results in 40--50 million dollars annual losses in profits to catfish producers. Early detection of this pathogen is necessary for disease control and reduction of economic loss. In this communication, the loop-mediated isothermal amplification method (LAMP) that amplifies DNA with high specificity and rapidity at an isothermal condition was evaluated for rapid detection of E. ictaluri. A set of four primers, two outer and two inner, was designed specifically to recognize the eip 18 gene of this pathogen. The LAMP reaction mix was optimized. Reaction temperature and time of the LAMP assay for the eip 18 gene were also optimized at 65 degrees C for 60 min, respectively. Our results show that the ladder-like pattern of bands sizes from 234 bp specifically to the E. ictaluri gene was amplified. The detection limit of this LAMP assay was about 20 colony forming units. In addition, this optimized LAMP assay was used to detect the E. ictaluri eip 18 gene in brains of experimentally challenged channel catfish. Thus, we concluded that the LAMP assay can potentially be used for rapid diagnosis in hatcheries and ponds.