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Development and Evaluation of a Loop-mediated Isothermal Amplification(LAMP) Assay for Rapid Detection of Chinese Giant Salamander Ranavirus 被引量:3
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作者 Yi GENG Xingxing LIU +5 位作者 Yan ZHOU Kaiyu WANG Xi PENG Zhijun ZHONG Xiaoli HUANG Defang CHEN 《Asian Herpetological Research》 SCIE CSCD 2015年第1期59-65,共7页
Loop-mediated isothermal ampliifcation (LAMP) is a novel nucleic acid diagnostic method that can amplify rapidly a target template under isothermal conditions. In this study, a LAMP assay for rapid detection of Chin... Loop-mediated isothermal ampliifcation (LAMP) is a novel nucleic acid diagnostic method that can amplify rapidly a target template under isothermal conditions. In this study, a LAMP assay for rapid detection of Chinese giant salamander ranavirus(CGSRV) was developed from culture isolates and clinical samples. The LAMP assay was developed by designing one set of four speciifc primers, targeting the major capsid protein (MCP) gene of CGSRV. Reaction time and temperature were optimal for 40 min at 62°C. The developed LAMP assay is speciifc and highly sensitive for CGSRV detection, the detection limit could reach about 5 copies of cloned viral genomic fragments. The sensitivity of the LAMP assay was about 1000 and 10-fold higher than that of both conventional and nested PCR, respectively. The LAMP ampliifcation produces a typical ladder-like pattern of products on an agarose gel that can be visually inspected after addition of ethidium bromide. The LAMP assay was evaluated further with clinical samples, and the results indicated the suitability and simplicity of the test as a rapid diagnostic tool for the detection of CGSRV. 展开更多
关键词 CGSRV loop-mediated isothermal ampliifcation lamp RANAVIRUS Chinese giant salamander
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The Development of a Loop-Mediated Isothermal Amplification (LAMP) Procedure for Plague Diagnostic 被引量:1
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作者 Mariana de Lira Nunes Carina Lucena Mendes-Marques +1 位作者 Alzira Maria Paiva de Almeida Nilma Cintra Leal 《American Journal of Analytical Chemistry》 2014年第16期1069-1077,共9页
Plague caused by Yersinia pestis is one of the infectious diseases subject to the International Health Regulations (IHR). Permanent monitoring of the focal plague areas is mandatory in order to enable prompt control m... Plague caused by Yersinia pestis is one of the infectious diseases subject to the International Health Regulations (IHR). Permanent monitoring of the focal plague areas is mandatory in order to enable prompt control measures to prevent the spread of the disease. Therefore, the availability of efficient diagnosis tests is of paramount importance. Here, we describe a loop-mediated isothermal amplification (LAMP)-based procedure for rapid Y. pestis detection. We constructed a set of LAMP primers, which were used in assays to establish the reaction conditions that would lead to the quick visualization of the results by evaluating the test tube with the naked eye. The primers were specifically designed to target the caf1 gene located on pFra/Tox (pMT), a prototypical plasmid of Y. pestis. The LAMP procedure was performed at 65&deg;C for 45 min in a water bath and allowed for the detection of at least 10 pg of bacterial DNA. Due to its simplicity, specificity, sensitivity and rapidity, the LAMP technique is an additional tool that may be implemented in routine plague diagnoses, especially in emergencies. 展开更多
关键词 PLAGUE YERSINIA PESTIS Diagnosis Tests loop-mediated isothermal amplification (lamp)
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Efficient detection of pathogen virus in sand dabs,Paralichthys olivaceus using loop-mediated isothermal amplification(LAMP) 被引量:1
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作者 HWANG Jinik PARK So Yun +3 位作者 SUH Sung-Suk PARK Mirye LEE Sukchan LEE Taek-Kyun 《Acta Oceanologica Sinica》 SCIE CAS CSCD 2016年第8期44-50,共7页
Viral hemorrhagic septicemia virus(VHSV) and marine birnavirus(MABV) are the causative pathogens for some of the most explosive epidemics of emerging viral diseases in many Asian countries, leading to huge economi... Viral hemorrhagic septicemia virus(VHSV) and marine birnavirus(MABV) are the causative pathogens for some of the most explosive epidemics of emerging viral diseases in many Asian countries, leading to huge economic losses in aquaculture. Rapid molecular detection for surveillance or diagnosis has been a critical component in reducing the prevalence of pathogen infection. The loop-mediated isothermal amplification(LAMP) of DNA is currently one of the most commonly used molecular diagnostic tools, as it is simple, quick, and easy to amplify target DNA under isothermal conditions. In the present study, a novel and highly specific LAMP assay for the sensitive and rapid detection of VHSV and MABV infection in fish was developed. Using a set of synthesized primers matching a specific region of the genome, the efficiency and specificity of the LAMP assay were optimized in terms of the reaction temperature and DNA polymerase concentration, as they are the main determinants of the sensitivity and specificity of the LAMP assay. In particular, we demonstrated that our assay could be applied to efficient detection of VHSV and MABV infection in the wild fish, Paralichthys olivaceus. Our results demonstrate the simplicity and convenience of this method for the detection of viral infection in aquatic organisms. 展开更多
关键词 viral hemorrhagic septicaemia virus(VHSV) marine birnavirus(MABV) polymerase chain reaction loop-mediated isothermal amplification
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Evaluation of Reverse Transcription Loop-Mediated Isothermal Amplification assays for Rapid Detection of Human Enterovirus 71 and Coxsackievirus A16 in Clinical Samples 被引量:9
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作者 Hong Zhang Kai Nie +8 位作者 Yunzhi Liu Le Luo Wei Huang Shuaifeng Zhou Mengjie Yang Yu Chen Jianmin Luo Lidong Gao Xuejun Ma 《Advances in Infectious Diseases》 2012年第4期110-118,共9页
A sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for human enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16) infection was further evaluated. The one step reaction was perfor... A sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for human enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16) infection was further evaluated. The one step reaction was performed in a single tube at 65?C for 45 min for EV71 and 35 min for CVA16. The detection limits of RT-LAMP assays for both EV71 and CVA16 were 0.1 of a 50% tissue culture infective dose (TCID50) per reaction, based on 10—Fold dilutions of a titrated EV71 or CVA16 strain. The specific assay showed there were no cross-reactions with Coxsackievirus A (CVA) viruses (CVA 2, 4, 5, 7, 9, 10, 14, and 25), Coxsackievirus B (CVB) viruses (CVB 1, 2, 3, 4, and 5) or ECHO viruses (ECHO 3, 6, 11, and 19). In parallel with commercial quantitative real-time polymerase chain reaction (qRT-PCR) diagnostic kits for EV71 and CVA16, the RT-LAMP assay was evaluated with 515 clinical specimens, the results showed the RT-LAMP assay and the qRT-PCR assay were in complete agreement for 513/515 (99.6%) of the specimens. Two samples with discrepant results from two methods were further verified by nested reverse transcription polymerase chain reaction (nRT-PCR) assay and sequencing to be true positives for CVA16. In conclusion, RT-LAMP assay is demonstrated to be a sensitive and specific assay and have a great potential for the rapid and visual screening of EV71 and CVA16 in China, especially in those resource-limited hospitals and rural clinics of provincial and municipal regions. 展开更多
关键词 Human ENTEROVIRUS 71 Coxsackievirus A16 REVERSE TRANSCRIPTION loop-mediated isothermal amplification
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Detection of coat protein gene of nervous necrosis virus using loopmediated isothermal amplification 被引量:2
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作者 Jinik Hwang Sung-Suk Suh +4 位作者 Mirye Park Myung-Joo Oh Jong-Oh Kim Sukchan Lee Taek-Kyun Lee 《Asian Pacific Journal of Tropical Medicine》 SCIE CAS 2016年第3期230-235,共6页
Objective:To establish a novel and highly specific loop-mediated isothermal amplification(LAMP) assay for the identification of nervous necrosis virus(NNV) infection.Methods:A set of synthesized primers was used to ma... Objective:To establish a novel and highly specific loop-mediated isothermal amplification(LAMP) assay for the identification of nervous necrosis virus(NNV) infection.Methods:A set of synthesized primers was used to match the sequences of a specific region of the nnr gene from the National Center for Biotechnology Information database,not originating from NNV-infected fish,the efficiency and specificity of LAMP were measured dependent on the concentration of DNA polymerase and the reaction temperature and time.In addition,to determine species-specific LAMP primers,cross reactivity testing was applied to the reaction between NVV and other virus families including viral hemorrhagic septicemia virus and marine birnavirus.Results:The optimized LAMP reaction carried out at 64 ℃ for 60 min,and above 4 U Bst DNA polymerase.The sensitivity of LAMP for the detection of nnv was thus about 10 times greater than the sensitivity of polymerase chain reaction.The LAMP assay primers were specific for the detection NNV infection in Epinephelus septemfasciatus.Conclusions:The development of LAMP primers based on genetic information from a public database,not virus-infected samples,may provide a very simple and convenient method to identify viral infection in aquatic organisms. 展开更多
关键词 Nervous NECROSIS VIRUS Nodaviridae POLYMERASE CHAIN reaction loop-mediated isothermal amplification
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Development of loop-mediated isothermal amplification for rapid detection of Entamoeba histolytica 被引量:2
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作者 Windell L.Rivera Vanissa A.Ong 《Asian Pacific Journal of Tropical Medicine》 SCIE CAS 2013年第6期457-461,共5页
Objective:To develop a loop-mediated isothermal amplification(LAMP) assay for the detection of Entamoeba histolytica(E.histolytica),the causative agent of amebiasis.Methods:The LAMP primer set was designed from E.hist... Objective:To develop a loop-mediated isothermal amplification(LAMP) assay for the detection of Entamoeba histolytica(E.histolytica),the causative agent of amebiasis.Methods:The LAMP primer set was designed from E.histolytica hemolysin gene HLY6.Genomic DNA of E.histolytica trophozoites strain HK9 was used to optimize the LAMP mixture and conditions.Amplification of DNA in the LAMP mixture was monitored through visual inspection for turbidity of the LAMP mix as well as addition of fluorescent dye.Results:Positive LAMP reactions turned turbid while negative ones remained clear.Upon addition of a fluorescent dye,all positive reactions turned green while the negative control remained orange under ambient light After elecrophoresis in 1.5% agarose gels,a ladder of multiple bands of different sizes can be oliserved in positive samples while no bands were detected in the negative control.The sensitivity of the assay was found to be S parasites per reaction which corresponds to approximately 1S.8 ng/μL DNA.The specificity of the assay was verified by the absence of amplified products when DNA from other gastrointestinal parasites such as the morphologically similar but non-pathogenic species,Entamoeba dispar. and other diarrhea-causing organisms such as Blastocystis hominis and Escherichia coli were used.Conclusions:The I.AMP assay we have developed enables the detection of E.histolytica with rapidity and ease,therefore rendering it is suitable for laboratory and field diagnosis of amebiasis. 展开更多
关键词 AMEBIASIS Diagnosis DNA ENTAMOEBA HISTOLYTICA HEMOLYSIN gene loop-mediated isothermal amplification assay
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Detection of Staphylococcus aureus in Dairy Food by Loop-mediated Isothermal Amplification 被引量:2
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作者 Cheng Xiao Wang Yongxin +4 位作者 An Hong Liu Juanjuan Zhang Bo Jia Zhen Cheng Jian 《Animal Husbandry and Feed Science》 CAS 2014年第3期127-130,共4页
[ Objective ] This study was to investigate the creditability of applying loop-mediated isothermal amplification method (LAMP) to detect Staphylococcus aureus in dairy products. [ Methods] The primers for heat resis... [ Objective ] This study was to investigate the creditability of applying loop-mediated isothermal amplification method (LAMP) to detect Staphylococcus aureus in dairy products. [ Methods] The primers for heat resistant nuclease gene (nuc) of Staphylococcus aureus were designed for establishing the LAMP method for rapidly detecting Staphylococcus aureus. [ Results ] The results of LAMP detection on Staphylococcus aureus in various dairy products were completely identical with that by bacterial isolation test; meanwhile it has a high specificity and a 10-fold sensitivity over the hemi-nested PCR. [ Conclusion] LAMP can be used for the detection of Staphylococcus aureus in dairy products. 展开更多
关键词 Staphylococcus aureus Nuc gene loop-mediated isothermal amplification
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Sensitive and rapid detection of two toxic microalgae Alexandrium by loop-mediated isothermal amplification 被引量:1
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作者 ZHANG Fengying SHI Yanhong +2 位作者 JIANG Keji XU Zhaoli MA Lingbo 《Acta Oceanologica Sinica》 SCIE CAS CSCD 2012年第2期139-146,共8页
A loop-mediated isothermal amplification (LAMP) assay was designed and evaluated for rapid de- tection of the toxic microalgae Alexandrium catenella and A. minutum, which can produce paralytic shellfish poisoning (... A loop-mediated isothermal amplification (LAMP) assay was designed and evaluated for rapid de- tection of the toxic microalgae Alexandrium catenella and A. minutum, which can produce paralytic shellfish poisoning (PSP). Two sets of four specific primers targeting these two species were derived from the sequence of internal transcribed spacer (ITS) of ribosomal DNA. The method worked well in less than an hour under isothermal conditions of 65℃. LAMP specificity was validated in closely related algae as a comparison, suggesting the strict specificity of the LAMP primers. Two visual inspection approaches were feasible to interpret the positive or negative results. The detection lim- its of A. catenella and A. minutum samples using the LAMP assay were found to be 5.6 and 4.5 pg DNA, respectively. The sensitivity of this LAMP assay was 10 or 100-fold higher than Polymerase Chain Reaction (PCR) method in detecting the two microalgae. These characteristics of species specificity, sensitivity, and rapidity suggest that this method has the potentiality in the monitoring of red tide caused by A. catenella and A. minutum. 展开更多
关键词 Alexandrium eatenella Alexandrium minutum detection loop-mediated isothermal amplification lamp ribosomal DNA internal transcribed spacer (ITS)
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Rapid detection of Shigella and Salmonella in rhesus monkeys by loop-mediated isothermal amplification assay 被引量:1
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作者 叶莉 吴芳 +6 位作者 WANG Yi-jia ZHENG Jun-wen FAN Jun-wen DING Ming SHI Yan-sheng 张小飞 白杰英 《中国实验动物学报》 CAS CSCD 北大核心 2016年第1期7-13,共7页
Objective To establish a loop-mediated isothermal amplification( LAMP) method for detecting diarrhea pathogens( Shigella and Salmonella) in rhesus monkeys and evaluate the application of the LAMP method for detecting ... Objective To establish a loop-mediated isothermal amplification( LAMP) method for detecting diarrhea pathogens( Shigella and Salmonella) in rhesus monkeys and evaluate the application of the LAMP method for detecting bacterial diseases in nonhuman primate laboratory animals. Materials and Methods A total of 205 fecal samples of rhesus monkeys were detected in this LAMP assay. The specificity and sensitivity of LAMP for Shigella and Salmonella were analyzed,and real-time polymerase chain reaction( REAL-TIME PCR) assay was employed as control. Results The LAMP method established here needed only 45 min to complete the reaction at 63℃. Its detection limit was 10 pg / μL and with a high specificity. The positive rate of Shigella and Salmonella was 1. 5% and 6. 3%,respectively. Conclusions Here we have established a fast and simple Shigella and Salmonella LAMP detection method that has strong specificity and high sensitivity and is suitable for rapid detection of bacterial disease in macaques. The development of this rapid detection kit is underway,and it will be helpful to the diarrhea detection. 展开更多
关键词 经典条件反射 操作式条件反射 SD大鼠 WISTAR大鼠 学习记忆
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Rapid,specific and sensitive detection of Vibrio vulnificus by loop-mediated isothermal amplification targeted to vvhA gene 被引量:1
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作者 ZHANG Lina WANG Mingyi +5 位作者 CONG Dianxia DING Shuyan CONG Rinan YUE Jinyong GENG Jianli HU Chengjin 《Acta Oceanologica Sinica》 SCIE CAS CSCD 2018年第4期83-88,共6页
Vibrio vulnificus is an estuarine bacterial pathogen for human.The rapid,specific and sensitive detection of V.vulnificus is urgently needed for early disease diagnosis and timely treatment of V.vulnificus infection.I... Vibrio vulnificus is an estuarine bacterial pathogen for human.The rapid,specific and sensitive detection of V.vulnificus is urgently needed for early disease diagnosis and timely treatment of V.vulnificus infection.In the study,a loop-mediated isothermal amplification(LAMP) technique was developed for V.vulnificus detection with a set of primers,composed of two out primers and two inner primers targeted to vvh A gene.The optimal amplification temperature was 63°C and the reaction only took 35 min.The amplification products could not only be detected by agarose gel electrophoresis with ladder-like pattern bands,but also could be visualized using calcein with naked eye directly.Forty-five strains were tested for the specificity of LAMP assay,and all the V.vulnificus strains were identified correctly while other strains were negative results.The sensitive of the new LAMP assay was 100-fold more sensitive than the conventional PCR.Meanwhile,all the V.vulnificus strains were detected correctly in spiked,clinical and environmental samples by the new LAMP assay.Compared with other well-known techniques,the new LAMP assay targeted to vvh A gene was extremely rapid,simple,sensitive and specific for V.vulnificus identification. 展开更多
关键词 Vibrio uulnificus loop-mediated isothermal amplification vvhA gene
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Rapid and Visualized Detection Method of Canine Parvovirus Using Loop-Mediated Isothermal Amplification
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作者 YANG Hui QU Guang-gang +1 位作者 ZHAO Yuan-kai SHEN Zhi-qiang 《Animal Husbandry and Feed Science》 CAS 2010年第1期34-37,共4页
[Objective] To develop a rapid and visualized detection method using loop-mediated isothermal amplification (LAMP), improve the detection rate of canine parvovirus (CPV) and reduce the testing cost. [ Method] Acco... [Objective] To develop a rapid and visualized detection method using loop-mediated isothermal amplification (LAMP), improve the detection rate of canine parvovirus (CPV) and reduce the testing cost. [ Method] According to the conserved regions of the VP2 gene of CPV, six primers were designed to amplify the special DNA sequences by LAMP. In addition, the reaction conditions of LAMP were optimized, and the sensitivity, specificity, repeatability and stability were verified. [ Result] The optimal reaction time of the LAMP method for CPVwas 60 min. The products obtained by LAMP had high specificity without cross-reaction with other generic viruses. The sensitivity of the LAMP was 100 times higher than that of PCR. [ Conclusion] The LAMP method for detecting CPV has high practical value. It has many advantages such as high specificity, high sensitivity, simple operation, low cost and rapid analysis, and it does not require special equipment. Therefore, this method is more suitable for the detection of CPV. 展开更多
关键词 Canine parvovirus loop-mediated isothermal amplification Diagnosis method
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Development of Reverse Transcriptase Loop-Mediated Isothermal Amplification for Rapid and Visualized Detection of Classical Swine Fever Virus
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作者 QU Guang-gang YANG Hui +5 位作者 TANG Na WANG Jin-liang FU Shi-jun XIE Jin-wen YUE Fu-jie SHEN Zhi-qiang 《Animal Husbandry and Feed Science》 CAS 2010年第3期21-24,共4页
[ Objective] To develop a rapid and visualized detection method of classical swine fever virus (CSFV) using reverse transcriptase loopmediated isothermal amplification (RT-LAMP). [ Method ] A total of six special ... [ Objective] To develop a rapid and visualized detection method of classical swine fever virus (CSFV) using reverse transcriptase loopmediated isothermal amplification (RT-LAMP). [ Method ] A total of six special primers were designed based on the conserved sequences of CSFV gene. After optimizing, the reaction of RT-LAMP was carded out at 63℃ for 45 rain. The RT-LAMP products were analyzed by agarose gel electro- phoresis. The sensitivity, specificity and repeatability were verified, respectively. [ Result] The RT-LAMP method could be used for detecting CSFV rather than six generic viruses. The sensitivity of RT-LAMP was 100 times higher than that of RT-PCR. The detection of 27 clinical samples by RT- LAMP and RT-PCR showed that RT-LAMP is more reliable and convenient. [ Conclusion] The RT-LAMP method is sensitive and reliable for the detection of CSFV. 展开更多
关键词 Classical swine fever virus Reverse transcriptase loop-mediated isothermal amplification RAPIDITY DIAGNOSIS
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猴痘病毒实时荧光LAMP检测方法的建立 被引量:2
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作者 许晓琳 袁向芬 +3 位作者 孔玉方 方鉴军 王慧煜 吴绍强 《黑龙江畜牧兽医》 CAS 北大核心 2024年第2期72-78,共7页
为建立一种特异性强、灵敏度高、操作简单的猴痘病毒(Monkeypox virus, MPXV)检测方法,试验针对MPXV保守区OPG002基因片段设计特异性引物,通过优化环介导等温扩增技术(loop-mediated isothermal amplification, LAMP)反应体系中内引物... 为建立一种特异性强、灵敏度高、操作简单的猴痘病毒(Monkeypox virus, MPXV)检测方法,试验针对MPXV保守区OPG002基因片段设计特异性引物,通过优化环介导等温扩增技术(loop-mediated isothermal amplification, LAMP)反应体系中内引物、环引物体积及反应温度建立了一种MPXV实时荧光LAMP检测方法,对该方法的特异性和灵敏度进行了分析,并应用该方法检测了临床样品和模拟样品。结果表明:建立的MPXV实时荧光LAMP检测方法于64℃恒温反应60 min即可完成DNA检测;该方法能特异性地检出MPXV,最低检出限为25 copies/μL,灵敏度是普通PCR方法的20倍;该方法对12份临床样品的检测结果均为阴性,可检测出质粒浓度为0.5×10~2~0.5×10~6 copies/μL的模拟阳性样品。说明建立的MPXV荧光LAMP检测方法具有特异性强、灵敏度高、操作简单的优点,可用于MPXV的快速鉴别诊断。 展开更多
关键词 猴痘 猴痘病毒 环介导等温扩增技术 特异性 灵敏度
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捻转血矛线虫LAMP-LFD检测方法的建立 被引量:1
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作者 段钰 汤洪宁 +4 位作者 项继忠 吴飞 马光旭 杜爱芳 杨怡 《动物医学进展》 北大核心 2024年第3期72-77,共6页
为了建立一种用于检测捻转血矛线虫的特异、灵敏、快速的检测方法,用环介导等温扩增技术(LAMP)结合横向流动试纸条(LFD),针对捻转血矛线虫mt-COⅠ基因的保守序列设计特异性引物和探针,对反应温度、反应时间、Mg^(2+)浓度、dNTP浓度进行... 为了建立一种用于检测捻转血矛线虫的特异、灵敏、快速的检测方法,用环介导等温扩增技术(LAMP)结合横向流动试纸条(LFD),针对捻转血矛线虫mt-COⅠ基因的保守序列设计特异性引物和探针,对反应温度、反应时间、Mg^(2+)浓度、dNTP浓度进行优化,建立了捻转血矛线虫环介导等温扩增技术与横向流动试纸条检测技术(LAMP-LFD)相结合的核酸检测方法。结果显示,建立的LAMP-LFD检测方法最低检测限为100 fg,对奥斯特线虫、夏伯特线虫、毛首线虫DNA检测均为阴性;50份临床样品检测结果与粪便虫卵检测法结果的符合率为96%。该方法操作方便、结果判定快速简单,有望成为流行区域和基层检测捻转血矛线虫的新方法。 展开更多
关键词 捻转血矛线虫 环介导等温扩增 横向流动试纸条 mt-COⅠ
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塞内卡病毒LAMP可视化快速检测方法的建立及初步应用
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作者 田瑶 李琛 +10 位作者 杨莹 李佳昕 王硕 时建立 彭喆 徐绍建 吴晓燕 刘畅 韩红 韩先杰 李俊 《中国动物传染病学报》 CAS 北大核心 2024年第3期88-94,共7页
为建立塞内卡病毒(SVA)的快速检测方法,本研究在SVA高度保守区域,分别设计了5对特异性LAMP引物,并通过反应条件优化、敏感性试验、特异性试验以及临床样本检测,建立了一种经济、特异、敏感的SVA检测方法。在62℃水浴55 min扩增出大于220... 为建立塞内卡病毒(SVA)的快速检测方法,本研究在SVA高度保守区域,分别设计了5对特异性LAMP引物,并通过反应条件优化、敏感性试验、特异性试验以及临床样本检测,建立了一种经济、特异、敏感的SVA检测方法。在62℃水浴55 min扩增出大于220 bp的特异性阶梯状条带,以质粒为模板进行敏感性试验结果表明该方法在5.1 copies/μL时仍可以检测出SVA,比普通PCR敏感1000倍。特异性试验结果显示该方法与猪肺炎支原体(MPH)、猪伪狂犬病病毒(PRV)、猪圆环病毒2型(PCV2)、猪圆环病毒3型(PCV3)、猪瘟病毒(CSFV)、猪繁殖与呼吸综合增病毒(PRRSV)均无交叉反应,应用该方法检测72例临床疑似病料,阳性率为33%(24例)。与普通PCR相比较,该方法操作简便、快速、特异、经济,本研究提供了一种更适于临床检测的SVA检测方法。 展开更多
关键词 塞内卡病毒 环介导等温扩增 快速检测
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小反刍兽疫LAMP反应结果可视化检测方法比较研究
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作者 索婧媛 郑茜之 +1 位作者 王博 刘学东 《野生动物学报》 北大核心 2024年第3期664-669,共6页
环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术是一种可应用于各种病原体检测、基因分型等领域的等温扩增技术。该技术所需设备简单且易于操作,因此具有较大的发展潜力。根据目前已经存在的判定LAMP结果的可视化方... 环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术是一种可应用于各种病原体检测、基因分型等领域的等温扩增技术。该技术所需设备简单且易于操作,因此具有较大的发展潜力。根据目前已经存在的判定LAMP结果的可视化方法,以可能对野生小反刍动物造成威胁的小反刍兽疫病毒(Peste des petits ruminants virus,PPRV)cDNA为检测对象,选择合适的引物组,比较评估均可在日光或紫外光下区分阴阳性结果的SYBR Green I、SYBR Safe Stain和羟基萘酚蓝(hydroxy naphthol blue,HNB)3种指示剂方法的适用性与灵敏度。结果表明:3种方法的灵敏度都很高,均与琼脂糖凝胶电泳相当。其中,SYBR Green I结果区分度高、不受体系成分影响且无须额外设备,但成本较高;SYBR Safe Stain具有良好的结果区分度,成本相对较低,但需要额外的紫外照射设备;HNB成本最低,但颜色区分度较低,且易受多种因素影响。建议在选择LAMP指示剂时,综合考虑成本、使用便利性、结果准确性及实验条件,通过充分的体系优化和验证,确保LAMP检测的准确性和可靠性。研究结果突显了不同方法的特征和适用场景,为研究人员根据不同场所与条件选择高效、便捷的LAMP反应结果可视化方法提供参考。 展开更多
关键词 环介导等温扩增 SYBR Green I SYBR Safe Stain 羟基萘酚蓝 小反刍兽疫病毒
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烟草番茄斑萎病毒RT-LAMP检测体系的建立及优化
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作者 张俊蕾 盖晓彤 +4 位作者 赵正婷 刘弟 王金凤 姜宁 刘雅婷 《中国农业科技导报》 CAS CSCD 北大核心 2024年第8期140-150,共11页
为快速检测烟草番茄斑萎病毒(tomato spotted wilt virus,TSWV),基于TSWV核外壳蛋白(nucleocapsid protein,NP)保守序列设计5组引物进行筛选,采用单一变量法对反应温度、时间及反应体系中dNTPs、Mg^(2+)、甜菜碱的含量和内外引物比等参... 为快速检测烟草番茄斑萎病毒(tomato spotted wilt virus,TSWV),基于TSWV核外壳蛋白(nucleocapsid protein,NP)保守序列设计5组引物进行筛选,采用单一变量法对反应温度、时间及反应体系中dNTPs、Mg^(2+)、甜菜碱的含量和内外引物比等参数进行优化,建立烟草TSWV反转录环介导等温扩增(loop-mediated isothermal amplification,RT-LAMP)检测体系,并采用RT-PCR(revers transcription-polymerase chain reaction)检测方法进行平行比对试验,验证优化后RT-LAMP的特异性、灵敏性及实用性。结果表明,烟草TSWV RT-LAMP检测体系最佳引物组为TS-N-4,在25μL的反应体系中各组分最佳加入量为缓冲液2.5μL、100 mmol·L^(-1) MgSO40.5μL、10 mmol·L^(-1) dNTPs 0.5μL、10 mmol·L^(-1) FIP/BIP 1.5μL、10 mmol·L^(-1) F3/B30.5μL、10 mmol·L^(-1) LF/LB 1.5μL、5 mmol·L^(-1) Betaine 6μL、Bst 2.0 WarmStar DNA Polymerase(8000 U·mL-1)0.5μL、M-MLV酶(10000 U·mL^(-1))0.125μL、RNA(≥64.7 fg)1μL,DEPC H2O补至25μL;最佳反应温度和时间分别为58℃、60 min。优化后的RT-LAMP灵敏度是RT-PCR的1000倍,且田间样品检测结果与RT-PCR相符。建立的RT-LAMP方法特异性强、灵敏度高、操作简单,对于烟草TSWV的检测及监控有重要意义。 展开更多
关键词 番茄斑萎病毒 NP基因 反转录-环介导等温扩增 快速检测
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华支睾吸虫LAMP方法的建立与初步应用
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作者 孙晓敬 马茜 +4 位作者 田甜 张磊 刘丽君 姚佳 汪洋 《黑龙江畜牧兽医》 CAS 北大核心 2024年第13期47-51,共5页
为了建立华支睾吸虫环介导等温扩增(loop-mediated isothermal amplification,LAMP)快速检测方法,试验利用GenBank中华支睾吸虫(Clonorchis sinensis)的线粒体全基因组设计特异性引物,然后构建LAMP方法,并验证了LAMP方法的特异性和敏感... 为了建立华支睾吸虫环介导等温扩增(loop-mediated isothermal amplification,LAMP)快速检测方法,试验利用GenBank中华支睾吸虫(Clonorchis sinensis)的线粒体全基因组设计特异性引物,然后构建LAMP方法,并验证了LAMP方法的特异性和敏感性;以及用粪便镜检(金标准)、PCR和LAMP方法同时检测45份犬粪便样本,评价LAMP方法的检测效果。结果表明:经序列比对,筛选得到华支睾吸虫特异性基因Unit R2,以该基因序列为靶标构建检测方法,优化后的总体积为50μL,Bst3.0 DNA聚合酶的最适用量为1μL,MgSO_(4)的最适浓度为6 mmol/L,最佳反应温度为63℃,最佳反应时间为40 min。构建的LAMP方法可以检测华支睾吸虫整个生命发育周期,实现了对其成虫、嚢蚴及虫卵的全方位覆盖;与其他种类寄生虫无交叉反应;对基因组DNA的最低检测限为10 fg,是PCR方法的100倍;与金标准相比,LAMP方法的特异性为100%(30/30),敏感性为93.33%(14/15)。说明试验建立的华支睾吸虫LAMP方法特异性强、敏感性高、检测结果准确,具有快速检测华支睾吸虫的潜力。 展开更多
关键词 华支睾吸虫 环介导等温扩增(lamp) 线粒体基因组 核酸 检测
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LAMP十三联检在COPD合并社区获得性肺炎患者中的应用价值
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作者 张兴 严秋梅 李玉英 《西部医学》 2024年第10期1546-1550,共5页
目的探讨环介导等温扩增技术(LAMP)十三联检在慢性阻塞性肺疾病(COPD)合并社区获得性肺炎(CAP)患者中的应用价值。方法回顾性分析2020年8月1日-2022年11月31日在西南医科大学附属医院呼吸与危重症医学科住院的COPD合并社区获得性肺炎患... 目的探讨环介导等温扩增技术(LAMP)十三联检在慢性阻塞性肺疾病(COPD)合并社区获得性肺炎(CAP)患者中的应用价值。方法回顾性分析2020年8月1日-2022年11月31日在西南医科大学附属医院呼吸与危重症医学科住院的COPD合并社区获得性肺炎患者343例,收集患者基本资料,留取患者入院后合格痰液标本,同时行痰细菌培养、LAMP十三联检,了解病原菌分布,并对比两种方法差异性及一致性。结果LAMP十三联检阳性率为49.85%,痰培养阳性率为20.99%,两种方法差异有统计学意义(χ^(2)=13.989,P<0.001)。一致性检验提示大肠埃希菌(Eco)一致性最好(Kappa值为0.796),鲍曼不动杆菌(Aba)、铜绿假单胞菌(Pae)、肺炎克雷伯菌(Kpn)、金黄色葡萄球菌(Sau)、耐甲氧西林葡萄球菌(Mrs)和嗜麦芽窄食单胞菌(Sm)Kappa值均>0.4,具有一致性,而流感嗜血杆菌(Hi)Kappa值仅为0.174,一致性差。COPD病程<10年组和≥10年组检出病原菌在Pae、单一感染和总阳性率差异有统计学意义(P<0.05);呼吸衰竭组和非呼吸衰竭组在Pae、单一感染、总阳性率差异有统计学意义(P<0.001)。结论LAMP十三联检能够快速、准确的识别病原体,有助于慢性阻塞性肺疾病合并社区获得性肺炎患者病原体的早期识别,对患者诊断与治疗有一定指导意义。 展开更多
关键词 环介导等温扩增技术 慢性阻塞性肺疾病 社区获得性肺炎 病原体
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响应面法优化LAMP反应冻干保护剂配方的研究
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作者 蔡文凯 祝珊珊 +2 位作者 郝宗杰 苏业俣 林艺平 《海南医学》 CAS 2024年第13期1889-1894,共6页
目的筛选出适用于甲流病毒环介导等温扩增技术(LAMP)的最佳冻干保护剂组合,建立起可在室温下长时间储存的甲流病毒核酸检测体系,为甲流现场快速检测和分级诊疗提供技术支持。方法以LAMP核酸扩增活性为评价指标,在单因素试验的基础上利... 目的筛选出适用于甲流病毒环介导等温扩增技术(LAMP)的最佳冻干保护剂组合,建立起可在室温下长时间储存的甲流病毒核酸检测体系,为甲流现场快速检测和分级诊疗提供技术支持。方法以LAMP核酸扩增活性为评价指标,在单因素试验的基础上利用三因素三水平的响应面法,探究冻干保护剂(海藻糖、牛血清白蛋白和甘露醇)对LAMP试剂的保护效果并优化冻干保护剂的组合。结果采用Box-Behnken试验结果进行回归模型拟合,LAMP试剂的保护剂最佳配方组合为7.64 wt%海藻糖、0.43 wt%牛血清白蛋白和0.85 wt%甘露醇。经真空冷冻干燥且加入保护剂后的LAMP试剂将以脱水形式贮存,可在常温下长期保持活性,使用时加水复溶即可。为了检验保护剂的稳定性,真空冷冻干燥后的LAMP试剂分别置于25℃和37℃下贮存,第0天、4天、8天、12天、16天、20天取出分别进行LAMP扩增活性检测。添加最优保护剂组合的LAMP冻干试剂其核酸扩增活性远大于添加单一保护剂或未添加冻干保护剂组。结论添加最优保护剂组合能够延长LAMP试剂的贮存时间和保持试剂的稳定性。 展开更多
关键词 响应面分析 环介导等温扩增技术 真空冷冻干燥 冻干保护剂
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