嗜水气单胞菌RPA-LFD快速检测方法的建立

文章旨在建立一种针对嗜水气单胞菌的敏感性高、特异性强的快速临床诊断方法。研究根据嗜水气单胞菌促旋酶B亚单位(gyrase subunit B, gyrB)基因的保守序列,设计特异性重组酶聚合酶扩增技术(RPA)扩增引物,通过结合琼脂糖凝胶电泳(AGE)和侧流层析试纸条(LFD)的方法进行反应条件的优化、特异性及灵敏度检测。实验结果表明,所建立的嗜水气单胞菌RPA-LFD快速检测方法可在38℃最适温度下30min内无干扰地检测到嗜水气单胞菌,对嗜水气单胞菌纯培养物和基因组DNA的最小检出限为10³ cfu/mL和100 pg/μL。利用建立的RPA-LFD与普通PCR同时检测杂交鲟鱼处理临床样品的结果表明,两种方法的结果一致。综上所述,通过对RPA反应条件的探索和优化,成功建立了嗜水气单胞菌快速检测方法。该方法操作简便,反应迅速,与普通PCR相比,不需要昂贵的仪器,为未来嗜水气单胞菌细菌性疾病的早期诊断提供了更有效的技术支持。     

重组酶聚合酶扩增技术研究进展

重组酶聚合酶扩增(recombinase polymerase amplification,RPA)技术是一种新兴的核酸恒温扩增技术,具有灵敏度高、特异性强、反应快速等特点。利用RPA技术可以对核酸拷贝数进行绝对定量并且可以同时检测多个靶标核酸序列,结合侧流层析试纸条或荧光信号监测装置等简易实验设备即可直接观察检测结果。就RPA技术的原理、发展、技术特点及其近年来在体外诊断、病原检测等领域的研究进展作一综述,旨在为该技术的深入研究和应用提供参考。     

LAMP-LFD技术及其在生物快检方面应用

LAMP-LFD技术是环介导横温扩增技术(Loop-mediated isothermal amplification)与横向流动试纸条(Lateral flow dipstick)相结合的一种新颖的检测技术,该技术将核酸横温扩增和可视化试纸条检测有机地结合。环介导等温扩增技术(LAMP),只需要在恒温(60℃~65℃)条件下保温几十分钟,即可将所需目的基因扩增到109水平。横向流动试纸条(LFD),融合了免疫层析技术和分子生物学手段,能在纸条上形成有颜色的检测线从而可以特异性地检测出该目标产物。环介导横温扩增技术(LAMP)与横向流动试纸条(LFD)相互结合的技术,使LAMP扩增产物现场检测可视化,检测结果明显直观,通过肉眼即可辨认,并具有高特异性、高灵敏度、简便、安全及成本低的特点,一度引起科研工作者的广泛关注。综述了环介导横温扩增方法与横向流动试纸条相结合(LAMP-LFD)技术的原理、优势、及其在生物快速检测方面的应用等,并指出了LAMP-LFD技术目前存在的不足以及展望。     

转基因玉米双抗12-5荧光RPA现场可视化检测方法的建立

转基因玉米双抗12-5具有良好的抗虫性和除草剂耐受性,是我国第一批获得安全证书的转基因玉米之一,具有广阔的应用前景。本研究利用重组酶聚合酶扩增技术(recombinase polymerase amplification, RPA)建立转基因玉米双抗12-5的现场快速检测方法。针对转基因玉米双抗12-5的转化体特异性序列片段,设计引物和探针,通过引物筛选实验得到最佳引物与探针组合。荧光RPA扩增结果可以在蓝光下直接进行可视化分析。结果表明,建立的转基因玉米双抗12-5可视化检测体系特异性强,检测灵敏度达到10拷贝。进一步研究发现RPA的扩增体系对温度有很强的适应性,样品在34℃～46℃之间都能得到扩增,据此,本研究利用市面上常见的自发热暖贴代替常规的加热仪器来激发RPA。结果表明,自发热暖贴满足RPA扩增体系对温度的需要。最终,本研究将自发热暖贴加热法与RPA可视化检测体系结合,对转基因玉米双抗12-5进行现场检测,并与qPCR方法检测结果作比较,检测结果表明,本研究建立的现场可视化检测方法与qPCR方法检测结果一致,并且可视化检测方法时间短,检测结果清晰易分辨。本研究建立的转基因玉米...     

重组酶聚合酶扩增技术研究进展

重组酶聚合酶扩增(RPA)是一种近年来发展起来的等温扩增技术。不仅具有检测速度快,灵敏度高,特异性好等特点,而且由于该反应可以在体温条件下等温扩增,特别适用于实验室外的现场诊断与床边诊断,具有广泛的应用前景。本文对RPA技术的发展进行了回顾与综述,对RPA技术的扩增原理、反应机制、反应体系、引物探针设计的优化,以及在此基础上发展起来的RPA产物检测方法,如侧向流试纸、絮凝分析、实时荧光、电化学、化学发光、表面增强拉曼散射等诊断技术进行了全面阐述,同时分析了当前RPA技术发展中的热点问题,为该技术的深入研究和推广应用提供参考。     

重组酶聚合酶扩增技术检测结核分枝杆菌

目的利用重组酶聚合酶扩增(Recombinase Polymerase Amplification,RPA)技术检测结核分支杆菌。方法采用Axxin T8-ISO扩增仪(TwistDX公司),反应温度设置为39℃,反应时间20min。检测分为实验组、阳性对照组和空白对照组。实验组模板DNA从痰液中提取。阳性对照模板DNA为H37Rv标准菌株样本DNA及卡介苗提取的DNA。空白对照为蒸馏水。结果 RPA技术可在20min内明显区分103～106 copies/mL的阳性质粒与阴性对照。对分离培养的结核杆菌(H37Rv)DNA及卡介苗提取的DNA,阳性克隆质粒进行等温扩增,结果结核杆菌分离株DNA、卡介苗DNA、阳性克隆质粒均有阳性扩增反应,而非结核分枝杆菌分离株和阴性对照无阳性扩增反应。灵敏度为100%,特异性为82%。结论 RPA技术利用了重组酶、单链结合蛋白、DNA聚合酶代替了传统PCR变性、退火、延伸的热循环过程,在常温25℃～42℃、15～30min内即可实现痕量核酸的快速扩增,可应用于快速检测人结核分枝杆菌,是一种全新的检测方法。     

重组酶聚合酶扩增技术在植物病毒检测中的应用

随着分子生物学技术的发展,多种核酸等温扩增技术逐渐被开发出来。其中,重组酶聚合酶扩增(recombinase polymerase amplification,RPA)作为一种快速、灵敏的检测技术具有很大的优势。目前,RPA已应用于转基因生物、各类病原物及食品安全检测等多个领域,并作为新兴技术在植物病毒检测领域中快速发展。RPA技术只需一对引物,在恒温条件下(37-42℃)只需30 min左右即可完成反应,具有较高的灵敏度与特异性。因此,该技术正迅速成为一种能够用于条件有限的实验室或现场植物病毒检测的手段。本文介绍了RPA技术的检测原理、引物设计和应用方式,综述了其在植物病毒检测中的最新研究进展及存在的问题,为RPA技术在植物病毒检测中的应用提供参考。     

环介导等温扩增联合横向流动试纸条快速检测创伤弧菌检测方法的建立

环介导等温扩增技术(LAMP)与横向流动试纸条(LFD)检测联合应用,建立了一种新的快速、便捷的创伤弧菌检测方法。针对创伤弧菌的外膜蛋白TolC基因设计6条特异性引物和1条异硫氰酸荧光素(FITC)标记的探针。生物素标记的LAMP扩增产物能够特异性地与FITC标记的探针杂交,杂交产物经LFD检测。优化后的扩增温度和时间为63℃反应35 min,加上细菌基因组DNA提取步骤,完成检测仅需要80 min。LAMP-LFD方法可特异性地检出创伤弧菌,对哈维氏弧菌等9种水产品常见病原菌的检测均呈阴性;对纯细菌培养物的检测灵敏度为3.7×102 CFU/mL或7.4 CFU/反应,是利用外引物建立的常规PCR检测的100倍。结果表明,该方法能够准确、快速、灵敏地检出创伤弧菌,可应用于创伤弧菌污染的水产品的检测。     

重组酶聚合酶扩增技术：一种新的核酸扩增策略

重组酶聚合酶扩增 (recombinase polymerase amplification, RPA)是近年来兴起的一种等温核酸扩增技术,它比聚合酶链式反应(polymerase chain reaction, PCR)及其它等温扩增技术更快速、便捷、高效。本文将详细介绍RPA这项新颖的技术,并对其在医疗诊断、农业、食品、生物安全等方面的研究及应用进展进行综述。期望这项技术得到更多的关注,使其发展更加完善,将来在更多的领域充分发挥作用,甚至书写核酸检测历史新篇章。     

一种可快速检测食品中酿脓链球菌和无乳链球菌的RPA方法的建立

为了实现食品中酿脓链球菌(Streptococcus pyogenes)和无乳链球菌(S.agalactiae)快速、高效检测,本研究建立了一种同时快速检测食品中这两种细菌的方法.本研究基于重组酶聚合酶等温扩增技术(recombinase polymerase amplification,RPA)原理,选择酿脓链球菌致...     

Rapid and sensitive detection method for Karlodinium veneficum by recombinase polymerase amplification coupled with lateral flow dipstick

The dinoflagellate Karlodinium veneficum that is usually present at relatively low cell abundances is a globally-distributed harmful algal bloom-forming species, which negatively affects marine ecosystems, fisheries, and human health. Hence, an efficient detection platform for the rapid and sensitive identification of K. veneficum is highly demanded. In this study, a method referred to as recombinase polymerase amplification coupled with lateral flow dipstick (RPA-LFD) was developed for the rapid detection of K. veneficum. The primers for RPA and the detection probe for LFD were designed to specially target the internal transcribed spacer of K. veneficum by molecular cloning and multiple alignments of the related sequences. The developed RPA can gain an approximately 300 bp specific band from K. veneficum. Successful amplification for RPA could be achieved at a temperature range of 35 °C–45 °C. RPA for 30 min could produce enough products that could generate clearly visible electrophoresis bands and were adequate for subsequent LFD analysis. The RPA products can be visually detected by the naked eyes through an LFD after an automatic chromatography for 5 min at room temperature. The developed RPA-LFD was exclusively specific for K. veneficum and displayed no cross-reactivity with other algal species that are commonly distributed along the Chinese coast. In addition, the lowest detection limit of RPA-LFD was 10 ng μL⁻¹ of genomic DNA and 0.1 cell mL⁻¹, which was 100-fold sensitive than conventional PCR. In conclusion, the developed RPA-LFD assay in this study can be used as a rapid and sensitive method to monitor K. veneficum in the future.     

Development of a recombinase polymerase amplification combined with a lateral flow dipstick assay for rapid detection of the <Emphasis Type="Italic">Mycoplasma bovis</Emphasis>

Background

Mycoplasma bovis (M. bovis) is a major etiological agent of bovine mycoplasmosis around the world. Point-of-care testing in the field is lacking owing to the requirement for a simple, robust field applicable test that does not require professional laboratory equipment. The recombinase polymerase amplification (RPA) technique has become a promising isothermal DNA amplify assay for use in rapid and low-resource diagnostics.

Results

Here, a method for specific detection of M. bovis DNA was established, which was RPA combined with lateral flow dipstick (LFD). First, the analytical specificity and sensitivity of the RPA primer and LF-probe sets were evaluated. The assay successfully detected M. bovis DNA in 30?min at 39 °C, with detection limit of 20 copies per reaction, which it was compared the real-time quantitative PCR (qPCR) assay. This method was specific because it did not detect a selection of other bacterial pathogens in cattle. Both qPCR and RPA-LFD assays were used to detect M. bovis 442 field samples from 42 different dairy farms in Shandong Province of China, also the established RPA-LFD assay obtained 99.00% sensitivity, 95.61% specificity, and 0.902 kappa coefficient compared with the qPCR.

Conclusions

To the author’s knowledge, this is the first report using an RPA-FLD assay to visualise and detect M. bovis. Comparative analysis with qPCR indicates the potential of this assay for rapid diagnosis of bovine mycoplasmosis in resource limited settings.

     

Concentrations of a Koi herpesvirus (KHV) in tissues of experimentally infected Cyprinus carpio koi as assessed by real-time TaqMan PCR

The Koi herpesvirus (KHV) is a herpes-like virus now recognized as a worldwide cause of mortality among populations of koi Cyprinus carpio koi and common carp Cyprinus carpio carpio. Temperature is a key factor influencing virus replication both in cell culture and in the tissues of experimentally infected fish. Genomic DNA sequences were used to optimize a rapid real-time TaqMan PCR assay to detect and quantify KHV DNA as found in the tissues of virus-exposed fish. The assay allowed analytical enumeration of target KHV genome copies ranging from 10(1) to 10(7) molecules as present in infected cell lines or fish tissues. The new assay was specific for KHV and did not detect DNA from 3 related herpes-like viruses found in fish, the Cyprinid herpesvirus 1 (CyHV-1), Cyprinid herpesvirus 2 (CyHV-2), Ictalurid herpesvirus 1 (IcHV-1) or the KF-1 cell line used for virus growth. Concentrations of KHV DNA were evaluated in 7 different tissues of replicate groups of virus-exposed koi held at water temperatures of 13, 18, 23 and 28 degrees C. Viral DNA was detected among virus-exposed koi at all 4 water temperatures but mortality was only observed among fish at 18, 23, and 28 degrees C. Time and temperature and the interactions between them affected concentrations of viral DNA detected in tissues of koi exposed to KHV. Although there were no recognized patterns to viral DNA concentrations as found in different tissues over time, KHV genome copies for all tissues increased with time post virus exposure and with water temperature. The remarkably rapid and systemic spread of the virus was demonstrated by the presence of viral DNA in multiple tissues 1 d post virus exposure. The greatest DNA concentrations found were in the gill, kidney and spleen, with virus genome equivalents consistently from 10(8) to 10(9) per 10(6) host cells. High levels of KHV DNA were also found in the mucus, liver, gut, and brain. Koi surviving infection at 62 to 64 d post virus exposure contained lower KHV genome copies (up to 1.99 x 10(2) per 10(6) host cells) as present in gill, kidney or brain tissues.     

Cloning of the koi herpesvirus (KHV) gene encoding thymidine kinase and its use for a highly sensitive PCR based diagnosis

Background  

Outbreaks with mass mortality among common carp Cyprinus carpio carpio and koi Cyprinus carpio koi have occurred worldwide since 1998. The herpes-like virus isolated from diseased fish is different from Herpesvirus cyprini and channel catfish virus and was accordingly designated koi herpesvirus (KHV). Diagnosis of KHV infection based on viral isolation and current PCR assays has a limited sensitivity and therefore new tools for the diagnosis of KHV infections are necessary.     

Initial characteristics of koi herpesvirus and development of a polymerase chain reaction assay to detect the virus in koi,Cyprinus carpio koi

Since 1998, episodes of mass mortality have occurred in populations of common carp Cyprinus carpio carpio in Israel and in populations of koi Cyprinus carpio koi in Israel and the USA. A herpesvirus isolated from infected fish has been shown in experimental studies to induce disease and mortality similar to those observed in outbreaks at infected farms. Initial characteristics of the virus show that it is clearly different from Herpesvirus cyprini (CHV), the most commonly known herpesvirus from cyprinid fish. The koi herpesvirus (KHV) has 31 virion polypeptides. Twelve of the virion polypeptides of KHV have similar molecular weights to those of CHV and 10 are similar to those of channel catfish virus (CCV). Both virion polypeptide and restriction fragment length polymorphism analyses of genomic DNA showed that the first KHV isolates from Israel and the USA were identical. In contrast, the genomic DNA restriction fragments clearly distinguish KHV from CHV and CCV. A polymerase chain reaction (PCR) assay to detect the virus in koi tissues was developed with sequences obtained from 1 restriction fragment of KHV DNA. The PCR assay effectively detected a 484 base pair sequence from KHV but did not amplify genomic DNA from either CHV or CCV. The PCR assay detected as little as 1 pg of KHV DNA mixed with 100 ng of host DNA. Viral sequences were amplified from koi obtained from field collections and from koi that were experimentally exposed to 10(2) TCID50 ml(-1) of KHV via the waterborne route. All KHV exposed fish dying of infection between 8 and 10 d post exposure or surviving to 14 d post exposure were found to be positive by PCR, while unexposed control koi were all negative. The assay also showed the presence of KHV DNA in tissues of koi obtained from farms in Israel. The PCR assay should assist virus isolation procedures and histologic and electron microscopic analyses now commonly used to detect KHV infection. Current studies are examining the possibility of using the PCR to detect KHV DNA in live fish and the relative sensitivity and specificity of the KHV PCR assay compared with other diagnostic tests.     

Koi herpesvirus disease

Koi herpesvirus (KHV) disease emerged at the late 1990s, and has rapidly spread to the world. In Japan, KHV disease first occurred at October 2003. The disease resulted in mass mortality of wild carp as well as cultured carp. Until now, KHV-infected carp were found in 42 out of 47 prefectures in Japan. Only carp Cyprinus carpio is susceptible to KHV, while goldfish, closely-related species to carp, is not. The affected carp swim lethargically. Sunken eyes and gill necrosis are frequently noticed, but no marked internal signs are observed. Optimal water temperature for the disease is 18-23 degrees C. Under 13 degrees C or over 28 degrees C, no death occurs. Keep at over 30 degrees C cures KHV disease, but can make the fish latent carriers. Because the fish do not get acquired immunity against KHV disease under low water temperature, the disease recurs with increase of water temperature. Isolation of KHV is difficult. KHV disease is diagnosed through epidemiological investigation, disease signs and PCR detection of KHV DNA. Vaccine development is ongoing for restart of culturing carp at KHV-contaminated places.     

Rapid Detection of Shrimp White Spot Syndrome Virus by Real Time,Isothermal Recombinase Polymerase Amplification Assay

White spot syndrome virus (WSSV) causes large economic losses to the shrimp aquaculture industry, and thus far there are no efficient therapeutic treatments available against this lethal virus. In this study, we present the development of a novel real time isothermal recombinase polymerase amplification (RPA) assay for WSSV detection on a small ESEQuant Tube Scanner device. The RPA sensitivity, specificity and rapidity were evaluated by using a plasmid standard as well as viral and shrimp genomic DNAs. Compared with qPCR, the RPA assay revealed more satisfactory performance. It reached a detection limit up to 10 molecules in 95% of cases as determined by probit analysis of 8 independent experiments within 6.41±0.17 min at 39°C. Consequently, this rapid RPA method has great application potential for field use or point of care diagnostics.     

Rapid detection of infectious bovine Rhinotracheitis virus using recombinase polymerase amplification assays

Background

Infectious bovine rhinotracheitis virus (IBRV) is a major pathogen in cattle and has led to significant economic losses to the dairy industry worldwide, and therefore a more optimal method for the rapid diagnosis of IBRV infection is highly needed. In this study, we described the development of a lateral flow dipstrip (LFD) of isothermal recombinase polymerase amplification (RPA) method for rapid detection of IBRV.

Methods

Distinct regions were selected as a candidate target for designing the LFD-RPA primers and probes. The analytical sensitivity of the RPA assay was determined using ten-fold serially diluted IBRV DNA. The specificity of the assay was assessed with other viral pathogens of cattle with similar clinic and other herpesviruses. The clinical performance was evaluated by testing 106 acute-phase high fever clinical specimens.

Results

RPA primers and probe were designed to target the specific conserved UL52 region fragment of IBRV. The detection could be completed at a constant temperature of 38 °C for 25 min, and the amplification products were easily visualized on a simple LFD. The detection limit of this assay was 5 copies per reaction of IBRV DNA and there was no cross-reactivity with other viruses causing bovine gastrointestinal and respiratory infections or other herpesviruses. The assay performance on acute-phase high fever clinical samples collected from cattle with no vaccine against IBRV, which were suspected to be infected with IBRV, was validated by detecting 24 fecal, 36 blood, 38 nasal swab and 8 tissue specimens, and compared with SYBR Green I based real-time PCR. The coincidence between IBRV LFD-RPA and real-time PCR was 100%.

Conclusion

IBRV LFD-RPA was fast and much easier to serve as an alternative to the common measures used for IBRV diagnosis, as there is reduction in the use of instruments for identification of the infected animals. In addition, this assay may be the potential candidate to be used as point-of-care diagnostics in the field.

     

Establishment and application of hyperbranched rolling circle amplification coupled with lateral flow dipstick for the sensitive detection of Karenia mikimotoi

The dinoflagellate Karenia mikimotoi is a noxious and harmful algal bloom (HAB)-forming microalga. Establishing a rapid, accurate, and sensitive method of detecting this harmful alga is necessary to provide warnings of imminent HABs through field monitoring. Here, an isothermal amplification technique combined with a rapid analytical method for nucleic acid-based amplified products, i.e., hyperbranched rolling circle amplification (HRCA) coupled with lateral flow dipstick (LFD), hereafter denoted as HRCA-LFD, was established to detect K. mikimotoi. The HRCA-LFD assay relied on a padlock probe (PLP) targeting DNA template and an LFD probe targeting PLP. The sequenced internal transcribed spacer of K. mikimotoi through molecular cloning was used as the target of PLP. The optimized HRCA conditions was determined to be as follows: PLP concentration, 20 pM; ligation temperature, 65 °C; ligation time, 10 min; amplification temperature, 61 °C; and amplification time, 30 min. The developed HRCA-LFD assay was specific for K. mikimotoi, displaying no cross-reactivity with other common microalgae. Sensitivity-comparison tests indicated that HRCA-LFD assay was 100-fold more sensitive than PCR, with a detection limit of 0.1 cell mL⁻¹ when used to analyze spiked field samples. The analysis with field samples also indicated that HRCA-LFD assay was suitable for samples with a target cell density range of 1–1000 cells mL⁻¹. All of these results suggested that HRCA-LFD assay is an alternative method for the sensitive and reliable detection of K. mikimotoi from marine water samples.