金黄葡萄球菌中毒休克综合征毒素1的研究

目的 应用PCR技术,检测金黄葡萄球菌的mecA基因和染色体上编码中毒休克综合征毒素1(TSST-1)的tst基因,了解tst基因的携带情况.方法 用PCR法对我院2006年8月-2007年5月临床分离的84株金黄葡萄球菌mecA基因和tst基因进行体外扩增,建立快速、特异、灵敏的检测产TSST-1耐甲氧西林金黄葡萄球菌(MRSA)的方法.结果 成功的对金黄葡萄球菌mecA基因和tst基因进行了检测,并进行基因测序.在我院84株受检金黄葡萄球菌中,41株金黄葡萄球菌的mecA基因扩增呈阳性,阳性株占48.81%.16株tst基因阳性,阳性株占19.05%.10株金黄葡萄球菌同时扩增出mecA基因和tst基因,阳性株占24.39%(10/41).结论 tst基因阳性株在临床分离的耐甲氧西林金黄葡萄球菌中占有较高的比例,应予以足够重视.     

人博卡病毒LAMP检测方法的建立及初步应用

目的 建立环介导等温扩增技术（LAMP）检测人博卡病毒（Human bocavirus,HBoV）基因的方法,并用于检测临床样本.方法 通过在线软件设计工具Ptimer Explorer V4设计HBoV NP-1基因的LAMP引物,建立LAMP检测方法,并评价其灵敏度和特异度.结果 与传统PCR方法相比,LAMP法检测灵敏度更高,可达到10拷贝,特异性较强;在76份呼吸道感染儿童的咽拭子标本中检测到5份阳性.结论 建立HBoV LAMP检测方法灵敏特异,简单快速,有望进行临床检测HBoV.     

基于环介导等温扩增的志贺菌快速检测方法的建立

目的 建立基于环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术的志贺菌快速检测方法.方法 针对志贺菌特异保守ipaH基因设计引物,通过引物筛选和反应条件优化,建立检测志贺菌的实时荧光及肉眼目视观测结果的LAMP方法,对方法的检出限、有无交叉反应、符合率和抗干扰...     

环媒恒温扩增(LAMP)技术在寄生虫检测中的应用

环媒恒温扩增(Loop-mediated isothermal amplification,LAMP)技术是一种快速、简单、敏感、特异的核酸扩增方法.作为一种新的核酸扩增方法,LAMP法被逐渐应用于寄生虫、病毒、微生物的检测,是最有发展前景的快速诊断方法之一,本文就LAMP技术在寄生虫检测方面的应用发展情况进行综述.     

LAMP和PCR法检测痢疾志贺菌的特异性和灵敏性比较

目的建立环介导等温扩增技术（LAMP）快速检测痢疾志贺菌,并对其特异性、灵敏度与传统PCR进行比较。方法以痢疾志贺菌侵袭性质粒抗原H基因（ipaH）为靶序列,设计6条特异性引物（内引物、外引物和环引物各2条）,优化LAMP反应体系及反应条件,检测LAMP反应的灵敏度、特异性及模拟样品,同步与PCR进行比较。结果 LAMP法和PCR均能特异性地扩增出志贺菌靶DNA,而其他非志贺菌均未扩增出特有条带。检测细菌纯培养物和模拟食品的灵敏度LAMP法分别为5.3×101cfu/ml、6.8×101cfu/ml,PCR法分别为5.3×102cfu/ml和6.8×102cfu/ml。结论利用LAMP技术,可快速、灵敏、简便地检测痢疾志贺菌,与PCR方法比较,特异性强、操作简便、检测成本低、耗时短,有望发展成为快速检测痢疾志贺菌的有效手段。     

环介导等温扩增技术检测间日疟原虫方法的建立及应用分析

目的建立一种快速、简便的检测间日疟原虫的环介导等温扩增方法（LAMP）并与常规PCR方法作比较。方法根据间日疟原虫环子孢子蛋白（CSP）基因序列合成2对特异性LAMP引物,优化Mg2＋浓度、dNTPs浓度、BstDNA聚合酶添加量、反应温度、时间以及设计引物缺省试验。评估优化后的LAMP反应的特异性和灵敏性。检测133份患者血样,以显微镜检方法为金标准,比较LAMP和多重PCR法检测间日疟原虫的敏感性和特异性。结果 LAMP法检测重组质粒DNA（Pv-rDNA）的灵敏度达到10-10,为传统PCR方法的100倍。镜检确诊的68例间日疟、43例恶性疟和22例非疟疾患者中,LAMP法和多重PCR检测间日疟原虫的敏感性为98.53%和97.06%,两法基本相当（χ2=0.34,P〉0.05）;特异性为86.15%和100%,LAMP法低于多重PCR法,差异有统计学意义（χ2=9.67,P〈0.05）。LAMP法的阳性预测值和阴性预测值分别为88.16%和98.25%,多重PCR的阳性预测值和阴性预测值分别为100%和97.01%。结论 LAMP法检测间日疟原虫具有快速简便、敏感性高、设备要求低的特点,具有较好的应用前景。     

环介导等温扩增技术可视化检测结核分枝杆菌

为了提升对结核分枝杆菌(MTB)检测的实用性,本文开展了采用环介导等温扩增方法(LAMP)对MTB进行可视化检测的研究。首先,根据MTB 16S r DNA序列设计LAMP引物。然后,收集临床痰液样本并进行相应处理。为了评价LAMP的特异度和灵敏度,本文用电泳产物进行检测并用钙黄绿素进行可视化验证。最后,以细菌培养结果为金标准,用SPSS 17.0软件比较培养结果与LAMP的一致性。结果显示,特异度实验中无非特异扩增现象,灵敏度实验中的检测极限为10拷贝。另外,可视化产物检测方法和电泳后的灵敏性相一致。进一步进行临床实用性评价,灵敏性为94.47%,特异性为90%,与细菌培养无统计学差异,结果一致性较好(P0.05)。综上,LAMP技术高效而可视,具有在设备匮乏的现场和基层医疗机构的应用前景。     

三种副溶血性弧菌分子检测试剂盒的评价

目的综合评价国内三种副溶血性弧菌商品试剂盒的检测效果。方法选用TIANDA公司的副溶血性弧菌PCR检测试剂盒、TAITAIGEN公司的副溶血性弧菌Real-timePCR检测试剂盒和HF公司的副溶血性弧菌LAMP检测试剂盒,以10倍稀释梯度的标准菌液确定各种试剂盒检出限;以国标法为标准,评价每种试剂盒的灵敏度、特异度和符合率;最后比较三种试剂盒的耗时、成本和操作性。结果 PCR、Real-timePCR和LAMP检测试剂盒的检出限分别为1.18×103cfu/ml、11.8cfu/ml和11.8cfu/ml;在74份海产品检测中,三种试剂盒的灵敏度分别为100%、100%和100%;特异度分别为56%、48%和50%;符合率分别为70%、65%和66%;三种试剂盒检测结果之间差异无统计学意义（P=0.074）;耗时分别为120、80和80min;成本分别为：10、40和45元/次;操作性从难到易排列依次为PCR、Real-timePCR和LAMP检测试剂盒。结论三种分子检测试剂盒的灵敏度高,特异度较低,操作简便,可推荐作为副溶血性弧菌检测的初筛方法。     

应用环介导等温扩增法快速检测O139群霍乱弧菌

目的 建立一种适合基层检验部门及小型实验室使用的快速检测O139群霍乱弧菌的方法.方法 应用环介导等温扩增技术(loop-mediated isothermal amplification,LAMP),针对O139霍乱弧菌wbfR基因设计4条引物(2条内引物、2条外引物);优化LAMP反应条件和反应体系,对反应体系中的引物、dNTP、Mg2+/Mn2+离子及Calcium等浓度进行优化;并对13株种系背景明确的霍乱弧菌不同实验对照株、30株O139群霍乱弧菌地方分离株、10株O1群霍乱弧菌地方分离株、32株其他肠道菌进行检测,验证该方法的特异性;通过肉眼目测或电泳检测比较结果.结果 所有O139群霍乱弧菌经LAMP榆测均呈绿色并电泳有阶梯状条带为阳性,O1群霍乱弧菌及其他肠道菌均检测呈橙色并电泳无相应条带为阴性;该体系最低检测限为63 CFU/反应;检测结果在白光下通过肉眼即可判断;从菌株核酸的提取至检测完成仅需1.5 h左右.结论 本研究建立的LAMP方法能够快速、灵敏、特异地检测O139群霍乱弧菌,无需昂贵的仪器,简单方便,非常适合基层检验部门或小型实验室以及流行病学人员于应急车上或现场监测等使用,值得推广.     

应用环介导等温扩增法快速检测O139群霍乱弧菌

目的 建立一种适合基层检验部门及小型实验室使用的快速检测O139群霍乱弧菌的方法.方法 应用环介导等温扩增技术(loop-mediated isothermal amplification,LAMP),针对O139霍乱弧菌wbfR基因设计4条引物(2条内引物、2条外引物);优化LAMP反应条件和反应体系,对反应体系中的引物、dNTP、Mg2+/Mn2+离子及Calcium等浓度进行优化;并对13株种系背景明确的霍乱弧菌不同实验对照株、30株O139群霍乱弧菌地方分离株、10株O1群霍乱弧菌地方分离株、32株其他肠道菌进行检测,验证该方法的特异性;通过肉眼目测或电泳检测比较结果.结果 所有O139群霍乱弧菌经LAMP榆测均呈绿色并电泳有阶梯状条带为阳性,O1群霍乱弧菌及其他肠道菌均检测呈橙色并电泳无相应条带为阴性;该体系最低检测限为63 CFU/反应;检测结果在白光下通过肉眼即可判断;从菌株核酸的提取至检测完成仅需1.5 h左右.结论 本研究建立的LAMP方法能够快速、灵敏、特异地检测O139群霍乱弧菌,无需昂贵的仪器,简单方便,非常适合基层检验部门或小型实验室以及流行病学人员于应急车上或现场监测等使用,值得推广.     

Detection of four Plasmodium species by genus- and species-specific loop-mediated isothermal amplification for clinical diagnosis

Loop-mediated isothermal amplification (LAMP), a novel nucleic acid amplification method, was developed for the clinical detection of four species of human malaria parasites: Plasmodium falciparum, P. vivax, P. malariae, and P. ovale. We evaluated the sensitivity and specificity of LAMP in comparison with the results of microscopic examination and nested PCR. LAMP showed a detection limit (analytical sensitivity) of 10 copies of the target 18S rRNA genes for P. malariae and P. ovale and 100 copies for the genus Plasmodium, P. falciparum, and P. vivax. LAMP detected malaria parasites in 67 of 68 microscopically positive blood samples (sensitivity, 98.5%) and 3 of 53 microscopically negative samples (specificity, 94.3%), in good agreement with the results of nested PCR. The LAMP reactions yielded results within about 26 min, on average, for detection of the genus Plasmodium, 32 min for P. falciparum, 31 min for P. vivax, 35 min for P. malariae, and 36 min for P. ovale. Accordingly, in comparison to the results obtained by microscopy, LAMP had a similar sensitivity and a greater specificity and LAMP yielded results similar to those of nested PCR in a shorter turnaround time. Because it can be performed with a simple technology, i.e., with heat-treated blood as the template, reaction in a water bath, and inspection of the results by the naked eye because of the use of a fluorescent dye, LAMP may provide a simple and reliable test for routine screening for malaria parasites in both clinical laboratories and malaria clinics in areas where malaria is endemic.     

Sensitive and rapid detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in shrimps by loop-mediated isothermal amplification

A method for nucleic acid amplification, loop-mediated isothermal amplification (LAMP) is a novel, sensitive and rapid technique, which can be applied for disease diagnosis in aquaculture. Using the LAMP method, a highly specific and sensitive diagnostic system for infectious hypodermal and hematopoietic necrosis virus (IHHNV) detection was designed. A set of four primers was designed by targeting the IHHNV genome DNA. By the detection system, target DNA was amplified and visualized on agarose gel within 60min under isothermal condition at 64 degrees C. Without gel electrophoresis, the LAMP amplicon was visualized directly in the reaction tube by addition of SYBR Green I for a naked-eye inspection. The LAMP reaction was also assessed by the white turbidity of magnesium pyrophosphate (a by-product of LAMP) in the tube. The assay had a detection limit of 5-500 copies of DNA template with gel electrophoresis, SYBR Green I and white turbidity with naked-eye inspection. The detection sensitivity of LAMP was 100-fold higher than the PCR. A diagnostic procedure which is rapid and highly sensitive was developed for IHHNV detection.     

Rapid detection of varicella-zoster virus infection by a loop-mediated isothermal amplification method

The reliability of varicella-zoster virus (VZV) loop-mediated isothermal amplification (LAMP) was evaluated for rapid diagnosis of viral infection. VZV-specific primers only amplified VZV DNA; no LAMP products were observed in reactions performed with other viral DNA templates. The specificity of this method was confirmed by two independent determinations, agarose gel electrophoresis and a turbidity assay. The sensitivity of VZV LAMP, determined by agarose gel electrophoresis, were 500 copies/tube. Detection using the turbidity assay, however, gave a sensitivity of 1,000 copies/tube. After these initial validation studies, reliability of VZV LAMP was evaluated for the detection of viral DNA in clinical specimens. Thirty-two swab samples collected from patients with vesicular skin eruptions were tested for VZV DNA. VZV was confirmed in sample numbers 10-32 by VZV real-time PCR, a previously established technique. VZV LAMP products were detected using turbidity from samples 13 to 32 (sensitivity; 87.0%, specificity; 100%, positive predictive value; 100%, negative predictive value; 75%). Although low levels of VZV DNA could be detected in the three samples exhibiting divergent results (samples numbers 10-12), no VZV LAMP product was detected in these samples, indicating a higher detection limit for this assay. Requirement of a DNA extraction step in the VZV LAMP method was examined in next experiment. The turbidity assay detected a VZV LAMP product in all of the 20 positive swab samples (samples numbers 13-32), regardless of DNA extraction.     

Detection of human herpesvirus 7 DNA by loop-mediated isothermal amplification

The reliability of loop-mediated isothermal amplification (LAMP), initially developed for the detection of human herpesvirus 7 (HHV-7), was evaluated in this study. Although a LAMP product was detected in HHV-7 DNA, neither HHV-6 nor human cytomegalovirus DNA produced a product. When agarose gel electrophoresis was used for the detection of LAMP products, the sensitivity of a 30-min HHV-7 LAMP reaction reached 250 copies/tube. The use of turbidity for the detection of the LAMP products gave a sensitivity of 500 and 250 copies/tube for 30- and 60-min reactions, respectively. Following these initial validation studies, clinical samples collected from two patients with primary HHV-7 infections were examined by HHV-7 LAMP. By use of agarose gel electrophoresis, HHV-7 LAMP products could be detected in acute-phase plasma samples but no LAMP product was detectable in convalescent-phase plasma samples from either patient. Since a turbidity assay is less sensitive than agarose gel electrophoresis, no HHV-7 LAMP product could be detected in plasma samples after a 30-min LAMP reaction. After a 60-min LAMP reaction, HHV-7 LAMP product could be detected in acute-phase plasma samples.     

环介导的等温扩增与实时荧光PCR检测问号钩端螺旋体的比较

目的 通过比较环介导的等温扩增技术(loop-mediated isothermal amplification,LAMP)与实时荧光PCR( real-time PCR)技术在检测问号钩端螺旋体的特异性及灵敏度方面的差异,寻找一种快速、灵敏且特异性强的问号钩端螺旋体检测方法。方法 根据问号钩端螺旋体lipL41基因序...     

Rapid and sensitive detection of Penaeus monodon nucleopolyhedrovirus by loop-mediated isothermal amplification

Loop-mediated isothermal amplification (LAMP) is a novel, sensitive and rapid method for amplification of nucleic acids under isothermal conditions. In this report, a LAMP method was developed for detection of Penaeus monodon nucleopolyhedrovirus (PemoNPV), known previously as monodon baculovirus (MBV), using a set of six primers designed to specifically recognize the PemoNPV polyhedrin gene. The optimized time and temperature conditions for the LAMP assay were 60 min at 63 °C. The sensitivity of LAMP for PemoNPV detection was approximately 50 viral copies ng⁻¹ genomic DNA (equivalent to 150 viral copies per reaction). Using a DNA template extracted from PemoNPV-infected shrimp by a viral nucleic acid kit, the detection limit of LAMP was 0.7 fg while that of nested PCR was 70 fg; therefore, the LAMP assay was 100 times more sensitive than nested PCR. The LAMP method did not amplify a product using nucleic acid extracted from shrimp infected with other viruses including yellow head virus (YHV), Taura syndrome virus (TSV), white spot syndrome virus (WSSV), Penaeus stylirostris densovirus (PstDNV) known previously as infectious hypodermal and hematopoietic necrosis virus (IHHNV), and Penaeus monodon densovirus (PmDNV) known previously as hepatopancreatic parvovirus (HPV).     

Direct detection of human herpesvirus 6 DNA in serum by the loop-mediated isothermal amplification method.

BACKGROUND: A more rapid and easier method is needed for monitoring human herpesvirus 6 (HHV-6) infections. The loop-mediated isothermal amplification method (LAMP) can detect viral DNA with high specificity, efficiency, and speed under isothermal conditions. LAMP requires only simple equipment that is available in hospital laboratories. OBJECTIVES: We evaluated LAMP as a means of detecting HHV-6 DNA directly from patients' sera. RESULTS: The sensitivity of the HHV-6 LAMP protocol without heat denaturation was 1000 copies/tube; with heat denaturation 10 copies/tube were detected. Three hundred serum samples from children with fever were analyzed. Using HHV-6 isolation as a definition of HHV-6 infection, the sensitivity, specificity, positive predictive value, and negative predictive value of the HHV-6 LAMP method without DNA extraction were 95.5%, 95.2%, 94.0%, and 96.4%, respectively. CONCLUSION: Direct detection of HHV-6 DNA in serum with a modified HHV-6 LAMP could be used for rapid diagnosis of exanthem subitum (ES).     

Loop-mediated isothermal amplification assay for the diagnosis of retinitis caused by herpes simplex virus-1

A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of herpes simplex virus 1 (HSV-1). The specificity of the assay was tested using DNA extracted from HSV-1-infected rabbit corneal epithelium cultures, HSV-2 grown on Vero cell line, cytomegalovirus (CMV) (AD-169), varicella zoster virus (VZV) (Oka-vaccine), adenovirus, Aspergillus flavus and Staphylococcus aureus. The specificity of LAMP was confirmed by bidirectional sequencing of the amplicons. The sensitivity of the LAMP assay was tested using different concentrations of HSV-1 DNA. To evaluate the application of the LAMP assay in clinical diagnosis, we tested vitreous samples from 20 patients with suspected viral retinitis using LAMP and real-time PCR for HSV-1. The LAMP primers amplified only HSV-1 DNA; no LAMP products were detected with the DNAs of HSV-2, CMV, VZV, adenovirus A. flavus and S. aureus. The sequences of the positive HSV-1 LAMP products perfectly (99–100%) matched the HSV-1 sequences deposited in the GenBank database. LAMP is as sensitive as real-time PCR, with the lowest detection limit being 10 copies/μL of HSV-1 DNA. Of the 20 patients with suspected viral retinitis, four tested positive for HSV-1 using real- time PCR and LAMP. A 100% concordance was observed across the two methods. The LAMP assay is a rapid, highly specific and sensitive method for the diagnosis of retinitis caused by HSV-1.     

Loop-mediated isothermal amplification assay for detection of Haemophilus influenzae type b in cerebrospinal fluid

Haemophilus influenzae type b (Hib) is one of the leading causes of meningitis in developing countries. To establish and evaluate a novel loop-mediated isothermal amplification (LAMP) assay for Hib, we designed a LAMP primer set targeting the Hib-specific capsulation locus. LAMP detected 10 copies of purified DNA in a 60-min reaction. This indicated that the detection limit of LAMP was >100-fold lower than the detection limits of both a PCR for the detection of bexA and a nested PCR for Hib (Hib PCR). No H. influenzae, other than Hib or control bacteria, was detected. Linear determination ranged from 10 to 1,000,000 microorganisms per reaction mixture using real-time turbidimetry. We evaluated the Hib LAMP assay using a set of 52 randomly selected cerebrospinal fluid (CSF) specimens obtained from children with suspected meningitis. For comparison, the CSF specimens were tested using a conventional Hib PCR assay. Hib was detected in 30 samples using LAMP and in 22 samples using the Hib PCR assay. The Hib PCR showed a clinical sensitivity of 73.3% and a clinical specificity of 100% relative to the Hib LAMP assay. These results suggest that further development and evaluation of the Hib LAMP will enhance the global diagnostic capability for Hib detection.