地黄花叶病毒RT-LAMP快速检测方法的建立及应用

基于反转录环介导等温扩增(reverse transcription loop-mediated isothermal amplification,RT-LAMP)技术建立了针对地黄花叶病毒(Rehmannia mosaic virus,ReMV)的快速、特异、灵敏的RT-LAMP检测方法。根据ReMV外壳蛋白(coat protein,CP)基因的核苷酸序列设计了3组LAMP引物,通过引物筛选确定最佳引物组合;利用电泳检测及可视化方法(加SYBR GreenⅠ显色剂)进行RT-LAMP温度优化、特异性检测、灵敏度比较。结果表明:本研究建立的RT-LAMP方法最适检测温度为60℃,优化后的LAMP方法能特异性检测ReMV,灵敏度是常规PCR的1000倍,可检测到1.2×10^(-1)拷贝/μL的模板浓度。对60份地黄样品的检测结果表明,RT-LAMP方法的阳性检出率为98.3%,高于常规RT-PCR方法(75%)。本研究建立的RT-LAMP检测技术为ReMV的准确检测提供了快速、高效的方法。

鸽新城疫病毒可视化LAMP检测方法的建立与应用

为建立一种简便、快速,适用于基层检测鸽Ⅰ型副黏病毒的方法,研究针对鸽Ⅰ型副黏病毒全基因组保守序列,设计了1组可检测基因Ⅵ、Ⅶ、ⅩⅩ、ⅩⅪ型分离株的环介导等温扩增(LAMP)引物,通过优化反应条件及体系,建立鸽Ⅰ型副黏病毒可视化LAMP检测方法。结果显示:建立的鸽Ⅰ型副黏病毒可视化LAMP检测方法可检测基因Ⅵ、Ⅶ、ⅩⅩ、ⅩⅪ型鸽新城疫病毒,与H9亚型禽流感病毒、马立克病病毒、禽网状内皮组织增殖病病毒、小鹅瘟病毒以及禽白血病病毒以及新城疫病毒La Sota疫苗株均无交叉反应,最低检测限为10^(1)copies/μL。研究表明所建立的鸽Ⅰ型副黏病毒可视化LAMP检测方法快速、简便,特异性强,灵敏度高,可应用于基层快速检测鸽Ⅰ型副黏病毒。

基于CP基因的云南烟草花叶病毒RT-LAMP快速检测体系的构建

为快速检测云南烟草花叶病毒(Tobacco mosaic virus,TMV),本研究根据来自云南烟草TMV分离物的外壳蛋白(CP)基因保守核苷酸序列,设计了5组引物进行筛选,并采用单一变量法对反应的温度、时间、甜菜碱浓度、dNTPs浓度、Mg2+浓度及内、外引物浓度比等进行逐一优化,建立了云南烟草TMV逆转录环介导等温扩增(RT-LAMP)检测体系。结果表明,最佳引物组为第4组,最适反应温度为60℃,甜菜碱、dNTPs、Mg^(2+)的最佳反应浓度分别为0.6、0.4、2.0 mmol/L,最佳内、外引物浓度比为4∶1,最佳反应时间40 min。优化后的RT-LAMP经SYBR Green I染色可肉眼判断结果,特异性高,灵敏度是常规RT-PCR的10倍。RT-LAMP检测体系的建立为云南烟草TMV的检测提供了一种便捷、高效、可靠的方法。

鸡圆圈病毒3型可视化LAMP检测方法的建立及应用

建立鸡圆圈病毒3型(GyV3)可视化环介导等温扩增(LAMP)检测方法,为临床上快速检测鸡GyV3提供一种诊断方法。参考GyV3的VP3基因序列,设计1套LAMP特异性引物,分别通过温度优化、引物浓度优化和反应时间的优化,确定鸡GyV3可视化LAMP诊断方法的扩增温度、引物浓度和反应时间,通过特异性试验、敏感性试验、重复性试验和临床样品检测,验证鸡GyV3可视化LAMP诊断方法的特异性、敏感性、稳定性和可靠性。结果显示,鸡GyV3 LAMP检测方法的反应体系:总体积为20.0μL,包括DNA模板1.0μL,2×Master Mix 10.0μL,内引物GyV3-FIP和GyV3-BIP混合液(工作浓度16.0μmol/L)2.0μL,外引物GyV3-F3和GyV3-B3混合液(工作浓度2.0μmol/L)2.0μL,环引物GyV3-LF和GyV3-LB混合液(工作浓度8.0μmol/L)2.0μL,ddH_(2)O 3μL;鸡GyV3 LAMP检测方法的扩增程序:66℃反应20 min,80℃灭活5 min。鸡GyV3可视化LAMP检测方法能特异性检测GyV3,最低检测浓度为10^(1)拷贝/μL,组内和组间变异系数小于5%;与常规PCR方法检测临床样品的结果比较,两者结果符合率达100%。建立的鸡GyV3可视化LAMP检测方法能特异性检测GyV3,具有特异性好、敏感性高及污染小等优点,且检测结果可通过肉眼观察进行判断,适用于GyV3的临床快速筛查。

华支睾吸虫LAMP方法的建立与初步应用

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。

虾类十足目虹彩病毒1(DIV1)LAMP-HNB快检方法的建立

[目的]建立一种操作简便、快速、可视化的虾类病原检测方法,实现基层养殖户自检。[方法]针对十足目虹彩病毒1(DIV1),设计环介导等温扩增(LAMP)特异性引物,对反应试剂用量、温度、反应时间等条件进行优化,建立LAMP与羟基萘酚蓝(HNB)结合的LAMP-HNB可视化快速检测方法。[结果]在LAMP反应体系中添加12 mmol/L Mg^(2+)和250μmol/L HNB,65℃恒温条件下反应60 min最佳;DIV1病原质粒最低检出限为102 copies/μL,稳定性和特异性较好,通过带有靶标基因的阳性质粒进行16次重复性试验结果均较为稳定,多种模板扩增试验均未发生假阳性情况,符合试验要求。[结论]该研究建立的LAMP-HNB可视化快速检测方法,适用于上述对虾DIV1的现场快速检测。

Security and Privacy in Solar Insecticidal Lamps Internet of Things:Requirements and Challenges

Solar insecticidal lamps(SIL) can effectively control pests and reduce the use of pesticides. Combining SIL and Internet of Things(IoT) has formed a new type of agricultural IoT,known as SIL-IoT, which can improve the effectiveness of migratory phototropic pest control. However, since the SIL is connected to the Internet, it is vulnerable to various security issues.These issues can lead to serious consequences, such as tampering with the parameters of SIL, illegally starting and stopping SIL,etc. In this paper, we describe the overall security requirements of SIL-IoT and present an extensive survey of security and privacy solutions for SIL-IoT. We investigate the background and logical architecture of SIL-IoT, discuss SIL-IoT security scenarios, and analyze potential attacks. Starting from the security requirements of SIL-IoT we divide them into six categories, namely privacy, authentication, confidentiality, access control, availability,and integrity. Next, we describe the SIL-IoT privacy and security solutions, as well as the blockchain-based solutions. Based on the current survey, we finally discuss the challenges and future research directions of SIL-IoT.

Observation of therapeutic effect of lamp irradiation combined with purple gromwell oil gauze on alleviating intestinal colic in patients

BACKGROUND Intestinal colic is a common complication in patients who have undergone radical surgery for colorectal cancer.Traditional Chinese medicine has advantages,including safety and stability,for the treatment of intestinal colic.Lamp irra-diation for abdominal ironing has been applied in the treatment of many gas-trointestinal diseases.Purple gromwell oil has the effects of clearing heat,cooling blood,reducing swelling,and relieving pain.RESULTS The general effective rate in the observation group was 95.00%,which was significantly higher than that in the control group(86.67%,P<0.05).Before treatment,there was no significant difference in the duration of symptoms between the groups(P>0.05).After 1,2,3,and 4 d of treatment,the duration of symptoms in both groups were decreased,and the duration in the observation group was significantly lower than that in the control group(96.54±9.57 vs 110.45±11.23,87.26±12.07 vs 104.44±11.68,80.45±16.21 vs 99.44±14.95,73.18±15.58 vs 92.17±14.20;P<0.05).After 1,3,5,and 7 d of treatment,the NRS scores in both groups were decreased,and the NRS scores in the observation group were significantly lower than those in the control group(3.56±0.41 vs 4.04±0.58,3.07±0.67 vs 3.74±1.02,2.52±0.76 vs 3.43±0.85,2.03±0.58 vs 3.03±0.82;P<0.05).There was no significant difference in the rate of adverse reaction occurrence between the groups(P>0.05).CONCLUSION The use of lamp irradiation combined with purple gromwell oil gauze in patients with intestinal colic after radical surgery for colorectal cancer can reduce symptom duration,alleviate intestinal colic,and improve treatment efficacy,and this approach is safe.It is worth promoting the use of this treatment in clinical practice.

Biomass Control via UV Lamp Application and Nutrient Supply Restriction in the Biofiltration of Gaseous VOCs

Biofiltration may have clogging problems owing to excess biomass growth during the treatment of gaseous pollutants.In this study,we employed an UV(Ultraviolet)lamp and controlled the nutrient supply to conduct a biofiltration process for treating 2-butanone(MEK:Methyl Ethyl Ketone)and toluene in a gas stream.Two methods of UV lamp usage(direct and indirect irradiation)and several nutrient supply methods were tested.However,no clear effect was observed with either UV usage.Under the optimal conditions,97%of the MEK and 69%of the toluene gases were removed after 29 s of EBRT(Empty Bed Retention Time).The inlet loads were 18 and 19 mg/(m^(3)·h)for MEK and toluene,respectively.Under these conditions,23 g-N/(m^(3)·day)of nitrate-nitrogen was consumed.Excess biomass growth occurred during simultaneous excess nutrient supply and a persistent irrigation schedule.In this study,we demonstrated the effective use of a dense nitrate solution to deliver an appropriate amount of nutrients and moisture,and the optimal irrigation frequency was four times per week.

实时荧光LAMP技术快速检测变形杆菌

建立一种快速检测变形杆菌的方法。采用实时荧光LAMP(Real Amp)技术检测变形杆菌,分析Gen Bank中公布的变形杆菌atp D基因序列,针对其6个区域设计4条引物,利用实时荧光监测仪等温(62℃)扩增模板DNA,通过与电脑相连进行实时监测,对该方法检测变形杆菌的特异性、灵敏度、人工污染猪肉检出限等方面进行研究,并将该检测法的灵敏度与普通环介导等温扩增(LAMP)检测法进行比较。结果表明,Real Amp检测方法比普通LAMP检测方法更加方便快捷,20 min内即可判定结果;用于特异性试验的19株试验菌株中,6株变形杆菌呈阳性结果,其他13株非变形杆菌均呈阴性结果;检测纯菌的灵敏度为8.1CFU/m L,是普通LAMP检测方法的10倍;人工污染猪肉的检出限为81CFU/m L。本研究建立的Real Amp检测方法操作简单,耗时短,特异性强、灵敏度高,能够实现对变形杆菌的快速检测。

传统方法、荧光定量PCR法、LAMP法检测痰液标本中肺炎链球菌的价值

目的:分析传统方法、荧光定量聚合酶链反应(PCR)法、环介导恒温扩增技术(LAMP)法检测痰液标本中肺炎链球菌的价值。方法:选取2022年1月—2023年7月医院收治的100例肺炎患者,均按相同方法严格采集患者的痰液标本后,采用痰培养法、荧光定量PCR法、LAMP法检测肺炎链球菌,分析不同检测方法的检测价值。结果:100例肺炎患者痰液标本经痰细菌培养法、荧光定量PCR法、LAPM法检测结果比较,差异无统计学意义(P>0.05),与痰细菌培养法相比,荧光定量PCR法、LAPM法对肺炎链球菌检测一致性高(Kappa=0.826、0.830,P<0.05)。以痰细菌培养法作为金标准,荧光定量PCR法、LAPM法对肺炎链球菌检测价值比较,无统计学差异(P>0.05)。结论:传统方法、荧光定量PCR法、LAMP法检测痰液标本中肺炎链球菌均有较高价值。

Design of Signal Lamp Filament Monitoring Alarm Instrument

To improve the reliability of the light emitting diode(LED)signal lamp filament current monitoring alarm instrument for metro systems,a new type of hot standby online monitoring apparatus was developed which is based on synchronous transmission data(STD)bus technology.In this system,a double hot standby mode can be achieved by adopting bus arbitration.In addition,to detect the effective value of alternating current which is from 0 to 200 mA in the signal lamp lighting circuit,a precision rectifier signal conditioning circuit and an isolated acquisition circuit were designed.This new type of alarm instrument has high detection accuracy and could meet the functional requirements for metro signal systems after comparing it with some industry products that were applied on the spot.

基于电化学DNA传感器的LAMP检测方法

申请号:202410558515.7【申请日】2024.05.08【公开号】CN118127133A【公开日】2024.06.04【分类号】G01N27/26;G01N27/48;C12Q1/6844【申请人】常州先趋医疗科技有限公司【发明人】关国良;王立新;贾瑶瑶【摘要】本发明属于电化学DNA传感器检测领域,具体提供了一种基于电化学DNA传感器的LAMP检测方法,包括:电化学DNA传感器制备;通过核酸外切酶Ⅲ对LAMP产物进行预处理;通过电化学DNA传感器制备对预处理后的LAMP产物进行电信号检测。应用核酸外切酶Ⅲ对LAMP扩增产物进行处理,使得LAMP产物中靶标区域单链DNA暴露出来,增强电化学DNA传感器检测的灵敏度,整个检测过程无需洗涤,避免了复杂的操作,减少误差。

草莓白化伴随病毒RT-LAMP检测方法的建立

草莓白化伴随病毒(strawberry pallidosis-associated virus,SPaV)能引起草莓白化病,与其他病毒复合侵染后会加重症状。本研究建立了SPaV的反转录环介导等温扩增方法(reverse transcription-loop-mediated isothermal amplification,RT-LAMP),对反应体系的各条件进行评估优化后,检测了RT-LAMP的特异性和灵敏度,且对田间样品进行了测试。结果表明,优化后的反应体系包含2.5μL 10×Isothermal Amplification Buffer、6 mmol/L Mg^(2+)、1 mmol/L dNTPs、1μmol/L SPaV-FIP和SPaV-BIP,0.1μmol/L SPaV-F3和SPaV-B3,0.3μmol/L SPaV-LB,0.4 mol/L Betaine,0.256 U/μL WarmStart Bst 2.0 DNA Polymerase,0.12 U/μL WarmStart RTx Reverse Transcriptase,1μL RNA,反应条件为61℃恒温孵育40 min。采用优化后的反应体系,在特异性检测中,只有携带了病毒SPaV的样品才能发生阳性扩增。灵敏度测试中,RT-LAMP比RT-PCR灵敏度高10倍。田间应用中,RT-LAMP和RT-PCR检测结果一致。本研究建立的SPaV RT-LAMP检测方法操作简便、快速、特异,可供实验室及生产实践中应用,助力脱毒种苗的鉴定和田间病毒病调查。

基于LAMP^(■)技术的核酸快速诊断技术平台建立

本文介绍了LAMP技术核酸快速诊断技术平台建立的流程,解决了LAMP法易污染、非特异扩增、无法定量、无法高通量等问题,并将RT-LAMP技术应用于H7N9疫情的检测,灵敏度能达到10 copy/μl,比RT-PCR高100倍,整个反应时间更短,比RT-PCR节约45min。LAMP法具有快速、恒温、简易、高特异、高敏感性的优点,特别适用于现场、基层医疗单位的快速检测。

Lamp Distribution Design for Adjustment of Florescence in Floral Production

[Objective] The aim was to introduce characters of light sources and select the optimal lamp scheme for floral production in greenhouses. [Method] With chrysanthemum cuttings as materials, HPSL, energy-saving fluorescent lamp, and LED agricultural lamp were made use of to carry out field tests in order to compare practical effects according to light characters of different lamps and plant growth de-mands. [Result] The results show that LED lamp performed the best of the three in practical use. LED square lamp designed in bat-wing shape would be the most ben-eficial for lamp distribution in a greenhouse. [Conclusion] LED agricultural lamp is the most popular currently, for it is energy saving and easy for operation.

小反刍兽疫LAMP反应结果可视化检测方法比较研究

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。

RT-LAMP与LAMP法检测结核分枝杆菌的效果比较

目的比较RT-LAMP、LAMP、L-J法检测MTB的效果,为结核病的快速诊断提供依据。方法用常见的非结核分枝杆菌与其他常见的呼吸道感染病菌(金黄色葡萄球菌、铜绿假单胞菌、肺炎克雷伯杆菌)检测特异性,并用酶切实验对结核分枝杆菌特异性产物进行酶切鉴定;用RT-LAMP、LAMP、L-J法检测100名结核病患者和22名来自无结核病患者痰标本中的结核分枝杆菌测试灵敏性;对浓度为1ng/μL H37Rv标准株进行对倍比稀释用于RT-LAMP极限检测实验。结果RT-LAMP、LAMP、L-J方法的阳性率分别为100%,92%和88%;RT-LAMP和L-J,RT-LAMP和LAMP差异均有统计学意义;RT-LAMP的灵敏度比LAMP高10倍;RT-LAMP不仅可以识别活菌,并且能够重复检测MTB的单一拷贝。结论 RTLAMP检测效果优于LAMP及L-J,适于基层医院结核病诊断的推广和应用。

猪细小病毒LAMP检测方法的建立

【目的】建立一种快速、敏感、特异的检测猪细小病毒(PPV)环介导等温扩增(LAMP)方法,为诊断猪细小病毒提供准确可靠工具。【方法】根据GenBank公布的PPV序列,在其保守序列区域设计了多套LAMP引物,利用LAMP Real Time Turbidimeter LA-320仪监测反应进程并筛选最佳引物、反应条件,建立了对PPV病毒DNA进行特异扩增的LAMP检测方法,并可通过加入SYBR Green I肉眼判断结果。【结果】该方法在63℃恒温下作用45min,PPV病毒DNA获得了高效率的特异性扩增;其检出限量为0.23TCID50,敏感性高;在反应结束后加入SYBR Green I肉眼判断结果,与Real Time Turbidimeter LA-320仪监测结果一致。通过对20份临床样品的LAMP检测与免疫荧光鉴定、PCR方法比对,符合率均为20/20。【结论】本研究建立的PPV LAMP检测方法具有快速、特异、灵敏,操作简单、设备要求低的特点,适合用于临床PPV快速检测。

鹅星状病毒LAMP快速检测方法的建立与应用

鹅星状病毒是一种新发的可导致雏鹅以痛风为主要特征的传染性病原。为建立鹅星状病毒(AstV)的快速检测方法,本研究根据鹅Ast V ORF1b基因的保守序列设计了一组特异性的LAMP引物,建立鹅AstV的LAMP快速检测方法,并检测其特异性和灵敏度。结果显示:建立的LAMP方法的最佳反应温度为64℃;仅能扩增鹅Ast V,而对鹅细小病毒、鹅副黏病毒、鸭坦布苏病毒及鹅源H9N2亚型禽流感病毒均不能扩增;能够检测到的最低模板(cDNA)浓度为1 ng/μL,因此该方法具有较好的特异性和敏感性。临床样品检测结果显示:建立的LAMP方法与RT-PCR方法阳性符合率为95.65%。综上,本研究建立的LAMP方法能够特异、快速地检测新发鹅Ast V,为养殖基地、科研和检验检疫部门提供了一种快速检测该病原的技术手段。