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。     

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。     

Rapid Detection of Mycobacterium tuberculosis by Recombinase Polymerase Amplification

Improved access to effective tests for diagnosing tuberculosis (TB) has been designated a public health priority by the World Health Organisation. In high burden TB countries nucleic acid based TB tests have been restricted to centralised laboratories and specialised research settings. Requirements such as a constant electrical supply, air conditioning and skilled, computer literate operators prevent implementation of such tests in many settings. Isothermal DNA amplification technologies permit the use of simpler, less energy intensive detection platforms more suited to low resource settings that allow the accurate diagnosis of a disease within a short timeframe. Recombinase Polymerase Amplification (RPA) is a rapid, low temperature isothermal DNA amplification reaction. We report here RPA-based detection of Mycobacterium tuberculosis complex (MTC) DNA in <20 minutes at 39°C. Assays for two MTC specific targets were investigated, IS6110 and IS1081. When testing purified MTC genomic DNA, limits of detection of 6.25 fg (IS6110) and 20 fg (IS1081)were consistently achieved. When testing a convenience sample of pulmonary specimens from suspected TB patients, RPA demonstrated superior accuracy to indirect fluorescence microscopy. Compared to culture, sensitivities for the IS1081 RPA and microscopy were 91.4% (95%CI: 85, 97.9) and 86.1% (95%CI: 78.1, 94.1) respectively (n = 71). Specificities were 100% and 88.6% (95% CI: 80.8, 96.1) respectively. For the IS6110 RPA and microscopy sensitivities of 87.5% (95%CI: 81.7, 93.2) and 70.8% (95%CI: 62.9, 78.7) were obtained (n = 90). Specificities were 95.4 (95% CI: 92.3,98.1) and 88% (95% CI: 83.6, 92.4) respectively. The superior specificity of RPA for detecting tuberculosis was due to the reduced ability of fluorescence microscopy to distinguish Mtb complex from other acid fast bacteria. The rapid nature of the RPA assay and its low energy requirement compared to other amplification technologies suggest RPA-based TB assays could be of use for integration into a point-of-care test for use in resource constrained settings.     

Non-Instrumented Incubation of a Recombinase Polymerase Amplification Assay for the Rapid and Sensitive Detection of Proviral HIV-1 DNA

Sensitive diagnostic tests for infectious diseases often employ nucleic acid amplification technologies (NAATs). However, most NAAT assays, including many isothermal amplification methods, require power-dependent instrumentation for incubation. For use in low resource settings (LRS), diagnostics that do not require consistent electricity supply would be ideal. Recombinase polymerase amplification (RPA) is an isothermal amplification technology that has been shown to typically work at temperatures ranging from 25–43°C, and does not require a stringent incubation temperature for optimal performance. Here we evaluate the ability to incubate an HIV-1 RPA assay, intended for use as an infant HIV diagnostic in LRS, at ambient temperatures or with a simple non-instrumented heat source. To determine the range of expected ambient temperatures in settings where an HIV-1 infant diagnostic would be of most use, a dataset of the seasonal range of daily temperatures in sub Saharan Africa was analyzed and revealed ambient temperatures as low as 10°C and rarely above 43°C. All 24 of 24 (100%) HIV-1 RPA reactions amplified when incubated for 20 minutes between 31°C and 43°C. The amplification from the HIV-1 RPA assay under investigation at temperatures was less consistent below 30°C. Thus, we developed a chemical heater to incubate HIV-1 RPA assays when ambient temperatures are between 10°C and 30°C. All 12/12 (100%) reactions amplified with chemical heat incubation from ambient temperatures of 15°C, 20°C, 25°C and 30°C. We also observed that incubation at 30 minutes improved assay performance at lower temperatures where detection was sporadic using 20 minutes incubation. We have demonstrated that incubation of the RPA HIV-1 assay via ambient temperatures or using chemical heaters yields similar results to using electrically powered devices. We propose that this RPA HIV-1 assay may not need dedicated equipment to be a highly sensitive tool to diagnose infant HIV-1 in LRS.     

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20 min。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20 min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℃、15~30 min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。     

Reverse Transcription Recombinase Polymerase Amplification Assays for Rapid Detection of Tick-Borne Encephalitis Virus Infection

<正>Dear Editor,Tick-borne encephalitis virus(TBEV) belongs to the genus Flavivirus within the family Flaviviridae and includes three subtypes: Siberian, European, and Far Eastern. TBEV infects humans via tick bites; indeed, at least 10,000 human cases of encephalitis caused by TBEV are reported annually in Russia, China, and European countries(Banzhoff     

Development of Recombinase Polymerase Amplification Assays for Detection of Orientia tsutsugamushi or Rickettsia typhi

Sensitive, specific and rapid diagnostic tests for the detection of Orientia tsutsugamushi (O. tsutsugamushi) and Rickettsia typhi (R. typhi), the causative agents of scrub typhus and murine typhus, respectively, are necessary to accurately and promptly diagnose patients and ensure that they receive proper treatment. Recombinase polymerase amplification (RPA) assays using a lateral flow test (RPA-nfo) and real-time fluorescent detection (RPA-exo) were developed targeting the 47-kDa gene of O. tsutsugamushi or 17 kDa gene of R. typhi. The RPA assay was capable of detecting O. tsutsugamushi or R. typhi at levels comparable to that of the quantitative PCR method. Both the RPA-nfo and RPA-exo methods performed similarly with regards to sensitivity when detecting the 17 kDa gene of R. typhi. On the contrary, RPA-exo performed better than RPA-nfo in detecting the 47 kDa gene of O. tsutsugamushi. The clinical performance of the O. tsutsugamushi RPA assay was evaluated using either human patient samples or infected mouse samples. Eight out of ten PCR confirmed positives were determined positive by RPA, and all PCR confirmed negative samples were negative by RPA. Similar results were obtained for R. typhi spiked patient sera. The assays were able to differentiate O. tsutsugamushi and R. typhi from other phylogenetically related bacteria as well as mouse and human DNA. Furthermore, the RPA-nfo reaction was completed in 20 minutes at 37^oC followed by a 10 minute incubation at room temperature for development of an immunochromatographic strip. The RPA-exo reaction was completed in 20 minutes at 39^oC. The implementation of a cross contamination proof cassette to detect the RPA-nfo fluorescent amplicons provided an alternative to regular lateral flow detection strips, which are more prone to cross contamination. The RPA assays provide a highly time-efficient, sensitive and specific alternative to other methods for diagnosing scrub typhus or murine typhus.     

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.     

实时荧光重组酶聚合酶扩增快速检测临床常见曲霉菌方法的建立

目的:针对曲霉菌属转录间隔区ITS1设计引物、探针,利用实时荧光重组酶聚合酶扩增(Real-time RPA)技术建立一种快速、准确、经济的临床常见曲霉菌检测鉴定方法。方法:利用建立的实时荧光重组酶聚合酶扩增体系对标准菌株及临床标本提取的DNA进行扩增,验证该方法的性能。结果:本研究针对曲霉菌属转录间隔区ITS1设计引物、探针利用RPA试剂盒(荧光型)建立了Real-time RPA扩增体系,在15分钟内即可检测出临床常见的四种曲霉菌;特异性试验结果显示反应体系只对烟曲霉、黄曲霉、土曲霉、黑曲霉四种曲霉呈现出明显的扩增曲线,而其它细菌和真菌均无扩增曲线。灵敏性试验显示最低检出限为10-3 ng/μL。临床验证试验的12份曲霉菌均有较高的扩增效应。结论:本研究建立的Real-time RPA方法能快速、特异、灵敏地检出烟曲霉、黄曲霉、土曲霉、黑曲霉等临床常见曲霉菌,为曲霉菌的快速、现场检测提供了一种新的思路。     

Development of a Lateral Flow Strip-Based Recombinase Polymerase Amplification Assay for the Detection of Haemonchus contortus in Goat Feces

Haemonchosis remains a significant problem in small ruminants. In this study, the assay of recombinase polymerase amplification (RPA) combined with the lateral flow strip (LFS-RPA) was established for the rapid detection of Haemonchus contortus in goat feces. The assay used primers and a probe targeting a specific sequence in the ITS-2 gene. We compared the performance of the LFS-RPA assay to a PCR assay. The LFS-RPA had a detection limit of 10 fg DNA, which was 10 times less compared to the lowest detection limit obtained by PCR. Out of 24 goat fecal samples, LFS-RPA assay detected H. contortus DNA with 95.8% sensitivity, compared to PCR, 79.1% sensitivity. LFS-RPA assay did not detect DNA from other related helminth species and demonstrated an adequate tolerance to inhibitors present in the goat feces. Taken together, our results suggest that LFS-RPA assay had a high diagnostic accuracy for the rapid detection of H. contortus and merits further evaluation.     

利用高保真酶扩增特性调整基因读码框的方法

在基因工程研究中,经常要涉及到对目的基因读码框进行调整的研究。本文报道了利用高保真DNA聚合酶扩增DNA后产生平末端的特性,及限制性内切酶对一重组的原核表达载体pET28a-HA进行读码框调整的研究,测序结果显示读码框按照预期设计正确调整,表明高保真DNA聚合酶可以用于DNA 3'凹陷端的补平,在DNA 3'凹陷端平滑化时多了一个有效的选择。     

A Fluorescent Recombinase Aided Amplification Assay for Detection of Babesia microti

Babesia microti is one of the most common causative agents of babesiosis. A sensitive and rapid detection is necessary for screening potentially infected individuals. In this study, B. microti cytochrome c oxidase subunit I (cox1) was selected as the target gene, multiple primers were designed, and optimized by a recombinase-aided amplification (RAA) assay. The optimal primers and probe were labeled with fluorescein. The sensitivity of fluorescent RAA (fRAA) was evaluated using gradient diluents of the cox1 recombinant plasmid and genomic DNA extracted from whole blood of B. microti infected mice. The specificity of fRAA was assessed by other transfusion transmitted parasites. The analytical sensitivity of the fRAA assay was 10 copies of recombinant plasmid per reaction and 10 fg/μl B. microti genomic DNA. No cross-reaction with any other blood-transmitted parasites was observed. Our results demonstrated that the fRAA assay would be rapid, sensitive, and specific for the detection of B. microti.     

利用反向PCR方法扩增细菌热激蛋白HSP60基因

利用PCR简并引物扩增出HSP6 0基因中一段约 6 0 0bp的核心片段 ,将该核心片段标记为探针 ,与基因组DNA进行Southern杂交 ,选择出适宜的限制性内切酶 ,以便消化基因组DNA得到大小合适的、含有HSP6 0基因的酶切片段。将酶切片段自身环化后作为模板进行反向PCR ,引物的延伸方向自核心片段出发延环化分子向未知序列区进行 ,可扩增出核心区上下游的序列。应用该方法 ,扩增并测定了寓齿双歧杆菌 (Bifidobacteriumdenticolens)DSM1 0 1 0 5 T、奇异双歧杆菌 (Bifidobacteriuminopinatum)DSM1 0 1 0 7T 和阴道加德纳氏菌 (Gard nerellavaginalis)ATCC1 40 1 8T 的HSP6 0全基因序列及青春双歧杆菌 (Bifidobacteriumadolescentis)JCM1 2 75 T98%以上的HSP6 0全基因序列。结果表明 ,反向PCR方法可有效的扩增细菌HSP6 0基因     

Diagnosis of Genital Herpes by Polymerase Chain Reaction Amplification

A new polymerase chain reaction (PCR) method employing type-specific primers and probes was applied to 114 clinical specimens obtained from 58 female patients with genital lesions or who had a history of genital herpes. Ten and 15 specimens, respectively, were positive for herpes simplex virus (HSV)-1 and HSV-2 by cell culture. All of 10 culture-confirmed HSV-1 cases and 11 of 15 (73%) culture-confirmed HSV-2 cases were identified by PCR. Although there were several cases with discrepancy between cell culture and PCR for HSV-2, the results suggest that this PCR procedure could be applied to clinical specimens from the female genital tract.