基于CRISPR/Cas蛋白的副溶血弧菌检测方法的研究

目的利用规律间隔性成簇短回文重复序列（CRISPR）免疫原理及Cas12a酶的特点构建一种快速检测副溶血弧菌（VP）的方法,实现对病原菌准确快速的检测和识别。方法本研究通过制备纯化Cas12a蛋白,筛选构建VP的gRNA,建立CRISPR-VP荧光检测系统,根据最终荧光扩增曲线判定CRISPR-VP检测方法的有效性。结果在CRISPR-VP检测方法中只在VP的序列存在时才会产生明显的荧光信号。结论本实验初步建立了基于CRISPR/Cas蛋白的VP的检测方法,为后续简易检测试剂的研制提供理论依据。     

CRISPR-Cas13a系统在基因编辑和分子诊断领域中的研究进展

成簇的规律间隔的短回文重复序列(CRISPR)-CRISPR相关蛋白(Cas)系统是一个强大的基因编辑工具。相对于Cas9,Cas13a可靶向多基因转录产物,从而调控基因功能表达,填补了Cas9仅限于DNA水平的编辑及脱靶效应等缺陷。不仅如此,利用Cas13a靶向RNA的特性,该系统被成功地改造成下一代核酸诊断工具。文章概述了CRISPR-Cas13系统在基因编辑及分子诊断领域的最新研究进展,并对该系统的应用前景进行了展望。     

核酸检测和基因编辑的新工具：CRISPR/Cas13系统

成簇的规律间隔的短回文重复序列(clustered regularly interspaced short palindromic repeats,CRISPR)/CRISPR相关蛋白(CRISPR-associated proteins, Cas)系统是目前基因编辑、基因表达研究的热点,其中,研究较成熟的为CRISPR/Cas9系统,其在单链RNA的引导下可特异地切割靶DNA的特定位点,实现DNA水平的操作。而靶向RNA的CRISPR/Cas13系统开启了在RNA水平研究、诊疗的新时代。活化的Cas13具有独特的核酸酶活性,可特异性地切割靶向RNA,同时非特异性地切割周围环境中的RNA,利用以上特性可实现体外核酸检测。通过活性位点突变可产生无核酸酶活性但可与RNA结合的dCas13(dead Cas13),将dCas13与其他功能性蛋白质进行融合可进一步扩大dCas13的应用范围。该文主要概括了CRISPR/Cas13系统在核酸检测以及在RNA水平作为基因编辑工具的新进展。     

CRISPR/Cas12系统在肿瘤检测中的应用

快速、灵敏、特异的检测方法对于提高肿瘤患者的生存率并改善预后至关重要。除了在基因编辑领域的贡献，近年来成簇规律间隔短回文重复序列(CRISPR)/CRISPR相关蛋白(Cas)系统以其特有的靶标核酸识别切割能力和反式切割活性为特点，已成为新一代核酸检测工具被广泛应用于病原体、肿瘤和转基因等检测领域。基于此，该文对CRISPR/Cas12系统原理及其在不同肿瘤标志物中的检测应用进展作一综述，并对其应用前景进行展望，以期为CRISPR/Cas系统更好地应用于肿瘤筛查和诊断提供参考及借鉴。     

副溶血弧菌的gyrB基因环介导的恒温扩增技术检测方法的研究

目的建立一种适合基层实验室应用和开展的快速检测副溶血弧菌的DNA环介导的恒温扩增法(LAMP)。方法针对副溶血弧菌的IgyrB/I 基因序列设计了4条引物（2条内引物、2条外引物）,并对扩增反应条件进行了优化。结果整个检测过程仅需1.5 h,可通过肉眼目测或电泳检 测判断结果;对3株种系背景明确的副溶血弧菌不同实验对照株、23株副溶血弧菌地方分离株和32株其他肠道菌进行了检测,具有很高的特异性 ;该方法的最低检测限为24 cfu/ml,具有良好的敏感性;应用于61份贝类海产品的现场检测,阳性率为100%,与实时荧光定量PCR方法的检测 结果相符,阳性率高于传统的培养鉴定方法。结论本方法具有快速、灵敏、特异、简便、经济等特点,适合基层实验室、应急检测或现场监测 等使用,具有较高的推广价值。     

规律成簇的间隔短回文重复序列中间隔序列的噬菌体来源研究

目的 发现在现有的全基因组测序完成的原核生物中规律成簇的间隔短回文重复序列(CRISPR)系统中间隔序列分布规律以及间隔序列中噬菌体来源情况. 方法 整理现有CRISPR数据库中2762株细菌基因组中的CRISPR系统和其中的间隔序列数据,整理GenBank数据库中发表的1444个噬菌体基因组数据.利用BLASTN软件对间隔序列数据与噬菌体基因组进行相似性比较,计数资料比较使用2检验. 结果 在2762个细菌基因组中整理出1940个基因组存在确定或可能的CRISPR结构和90 096条间隔序列,多数基因组具有1~50条间隔序列(1414/1940,72.9%),间隔序列数量250条的仅有58个基因组(58/1940,3.0%).其中古细菌13株(13/150,8.6%),真细菌45株(45/2612,1.7%),差异有统计学意义(2=29.98,P 0.01).相似性比较结果共发现245个细菌基因组的1055条间隔序列,成功比对上363个噬菌体,比对成功率仅为0.12%. 结论 细菌基因组中的CRISPR系统中间隔序列数量存在较大差异,古细菌基因组中CRISPR系统存在更多的间隔序列.相似性比较中噬菌体来源的间隔序列所占比例低,提示与细菌和噬菌体基因组发现较少相关,进一步深入研究可以大幅度提高成功率.     

副溶血弧菌的致病性及检测方法

副溶血弧菌是沿海地区的一种嗜盐性细菌,其生长快,繁殖速度高,常通过感染多种海产品而引发人的食物中毒发生,近年来,由副溶血弧菌引起的食源性疾病日益增多,到目前为止,O3：K6型副溶血弧菌菌株已引起了亚洲、欧洲、美洲、非洲等多个国家的大流行。随着由副溶血弧菌引起疾病的规模不断增加和频发,人们对其也更加关注,各种检测方法逐渐取代传统的生化鉴定实验,尤其是在食品微生物检测、流行病学调查以及医院感染监控等方面发挥越来越重要的作用。本文就副溶血弧菌的基础特性。致病性及目前的检测方法作一综述。     

基于CRISPR/Cas技术的核酸即时检测研究进展

鉴于目前核酸分子检测技术逐步从临床实验室向现场检测转移的发展趋势,急需建立一种高灵敏、高特异、高效和便捷的新型核酸诊断工具来满足临床即时检测（POCT）的需要。基于规律间隔成簇短回文重复序列（CRISPR）/CRISPR相关蛋白（Cas）的检测方法是一种有前景的新型核酸检测方法。该方法中Cas效应蛋白能够识别高度特异的...     

CRISPR/Cas系统在分子检测中的应用

近年来,成簇规律间隔短回文重复序列/成簇规律短回文重复序列相关蛋白(CRISPR/Cas)系统凭借其简单、高效的基因编辑能力,已被广泛应用于生物、医学等多个研究领域。随着CRISPR技术的快速发展,CRISPR/Cas系统已被开发为一种快速、便携、低成本、高灵敏度的分子检测工具,在病原体检测、耐药性分析、单核苷酸多态性(SNP)分型、肿瘤基因突变检测等方面取得重大突破。文章就不同Cas蛋白在分子检测中的最新研究进展进行综述,并对其应用前景进行展望,以期为从事相关领域的科研工作者提供参考与帮助。     

一起副溶血弧菌引起的食物中毒调查及检测

2009年10月5日下午17时左右,我单位接到辖区内某医院急诊科报告,该科收治了一批因腹痛、腹泻、发热等症状前来就诊的病人,怀疑是食物中毒。经流行病学调查、临床诊断和病原学鉴定,判定为一起由副溶血弧菌引起的食物中毒事件,现报告如下。1材料与方法1.1患者临床症状22例患者均有不同程度的腹痛、腹泻、恶心、呕吐、头晕     

Development of a rapid detection method to detect tdh gene in Vibrio parahaemolyticus using 2-step ultrarapid real-time polymerase chain reaction

Thermostable direct hemolysin encoded by tdh gene has been considered an important virulence factor in pathogenic Vibrio parahaemolyticus. Two-step ultrarapid real-time polymerase chain reaction (URRT PCR) with a microchip was devised to detect V. parahaemolyticus carrying tdh gene. This novel method has a 6-μL reaction volume and extremely reduces running time since one cycle can be completed in 10 s or less. Consequently, 35 cycles of URRT PCR was successfully able to detect up to 100 fg (18 copies) of genomic DNA from pathogenic V. parahaemolyticus carrying tdh gene in 6 min. These results indicate that this method is at present the most rapid detection method for tdh gene and pathogenic V. parahaemolyticus.     

Multiplex PCR for the detection and differentiation of Vibrio parahaemolyticus strains using the groEL,tdh and trh genes

Vibrio parahaemolyticus is a significant cause of human gastrointestinal disorders worldwide, transmitted primarily by ingestion of raw or undercooked contaminated seafood. In this study, a multiplex PCR assay for the detection and differentiation of V. parahaemolyticus strains was developed using primer sets for a species-specific marker, groEL, and two virulence markers, tdh and trh.Multiplex PCR conditions were standardised, and extracted genomic DNA of 70 V. parahaemolyticus strains was used for identification. The sensitivity and efficacy of this method were validated using artificially inoculated shellfish and seawater. The expected sizes of amplicons were 510 bp, 382 bp, and 171 bp for groEL, tdh and trh, respectively. PCR products were sufficiently different in size, and the detection limits of the multiplex PCR for groEL, tdh and trh were each 200 pg DNA. Specific detection and differentiation of virulent from non-virulent strains in shellfish homogenates and seawater was also possible after artificial inoculation with various V. parahaemolyticus strains.This newly developed multiplex PCR is a rapid assay for detection and differentiation of pathogenic V. parahaemolyticus strains, and could be used to prevent disease outbreaks and protect public health by helping the seafood industry maintain a safe shellfish supply.     

三起食物中毒来源副溶血弧菌毒力基因检测分析

目的分离3起食物中毒患者粪便及食物加工工具样本中的病原菌,对分离菌株进行表型和毒力基因鉴定。方法可疑病原菌的分离与鉴定按照国标GB/T 4789-2003和GB/T 4789.7-2008规定的方法进行;用PCR方法对检出的副溶血弧菌(IVibrioparahaemolyticus,/IVp)进行Vp种属特异基因(320 bp)和毒力基因Itdh/I( 耐热溶血素434 bp)、Itrh/I( 耐热相关溶血素250 bp) 检测;用环介导恒温扩增(LAMP)方法对分离株Itlh/I( 不耐热溶血素229 bp)基因进行检测。结果从3起食物中毒的患者、厨师粪便和食品加工环节共27件样品中检出7株Vp,其中加工环节熟食冷菜间刀具涂抹物中检出1株,患者粪便中检出6株。3起食物中毒7株菌血清型分属为O10∶K66、O2∶K3、O1∶K33, 每起食物中毒分离株的生物学性状、血清型和药敏试验结果均一致。7株Vp均产生神奈川现象（KP）、出现相同Vp种属特异基因条带、均检出Itlh/I毒力基因,但Itdh/I、Itrh/I基因则检测为阴性。结论3起食物中毒均为Itlh/I+/Itdh/I-/Itrh/I-不同血清型Vp污染引起。     

Non-isotopic microtitre plate-based assay for detecting products of polymerase chain reaction amplification: application to detection of the tdh gene of Vibrio parahaemolyticus.

A non-isotopic microtitre plate-based assay method was devised for detection of products of the polymerase chain reaction. This assay involves affinity immobilization of the biotinylated amplification products in microtitre plate wells and their fluorescence detection by their hybridization with an oligonucleotide probe linked to alkaline phosphatase. An advantage of this procedure is that the immobilization and hybridization are carried out simultaneously in the wells, thus shortening the assay time. The assay method was applied to the detection of the tdh gene of Vibrio parahaemolyticus. Seven copies of the target chromosome could be detected in about 45 min after 35 cycles of amplification.     

副溶血性弧菌快速检测方法研究

目的 以检测toxR基因作为副溶血性弧菌种水平上的特异性基因鉴定,建立一种快速、准确、简便的副溶血性弧菌(VP)筛选方法.方法 根据toxR基因序列设计合成引物,扩增被检VP所含的相应基因.分别用标准株及地方株做对照,同时与常规鉴定方法比较.结果 对浙江省2005年收集的286株来自临床病人和海产品的可疑VP鉴定,用常规方法鉴定出VP占总数的62.24%;用toxR基因检测,鉴定率占所有菌株的61.54%;两者差异无统计学意义(x2=0.03,P＞0.05),对40份海产品增菌液直接检测toxR基因与常规增菌分离鉴定法,均有37份标本检出VP,检出率为92.5%.结论 该检测方法快捷、特异性好、敏感性高,可以作为VP快速筛选方法.     

Coupling nucleic acid circuitry with the CRISPR-Cas12a system for universal and signal-on detection

We report a universal and signal-on HCR based detection platform via innovatively coupling the CRISPR-Cas12a system with HCR. By using this CRISPR-HCR pathway, we can detect different targets by only changing the crRNA. The CRISPR-HCR platform coupling with an upstream amplifier can achieve a practical sensitivity as low as ∼aM of ASFV gene in serum.

We report a universal and signal-on HCR based detection platform via coupling the CRISPR-Cas12a system with HCR. By using this CRISPR-HCR pathway, we can detect different targets by only changing the crRNA, with practical sensitivity as low as ∼aM.

A series of non-enzymatic isothermal nucleic acid circuiting reactions, known as enzyme-free DNA circuits,^1–4 have caught increasing attention because they are able to execute nucleic acid assembly with low cost, high flexibility, programmability, and readout comparability. Hybridization chain reaction (HCR), first reported by the Piece group,⁵ is one of the most important examples of enzyme-free DNA circuits.^6–8 It relates to a pair of complementary DNA hairpins with sticky ends (H1, H2) and an initiator (I₀). During the pathway, the pair of complementary DNA hairpins is utilized as fuel packets to achieve self-assembly, forming a long-nicked double-stranded DNA (dsDNA) polymer. Due to the reaction can provide both size and concentration amplification, HCR has been deep-studied and extensively-utilized as an intelligent sensing unit during molecular recognition, amplification, and transduction. The reactions have been engineered and designed for various different targets, such as nucleic acids,^9,10 proteins,¹¹ small molecules,¹² ions,¹³ bacteria,¹⁴ cells,¹⁵etc.¹⁶However, there are still problems in the existing HCR technology, which often limit its practical applications. For example, how to export an effective signal for an HCR reaction or products is always a serious challenge. The most-used signal is fluorescence resonance energy transfer (FRET).¹⁷ It is a common agreement that such a “signal-off” response may get easily confused with the false positives and insufficient intuitiveness. Therefore, continuous efforts have been made to develop “signal-on” responses. In many cases, classic HCR hairpin substrates have to be extended with additional oligonucleotides (named tails) to form an assembly-probe that can generate downstream signals.^{6–8,18–21} Taking an example from our previous publications,²² after HCR two adjacent tails can integrate into a full G-quadruplex structure. A porphyrin derivative (abbreviated as PPIX) can insert into the G-quadruplex and get much enhanced fluorescence emission. By similar concept, many other readouts, such as colorimetric, luminescent, and enzymatic signals have also been successfully realized.^23,24 Even though, experimental evidence has revealed once the HCR hairpin is added with additional tail sequences, the assembly efficiency or signal-to-background ratio may be seriously exhibited, which makes the HCR difficult to control, design, and apply.²² In addition, because the HCR initiator is always the target itself or a sequence highly relevant to the target, the hairpin sequences have to be re-built, re-designed, and/or re-optimized once a new target is detected, adding the time, cost, and risk for failure.Above issues are common for most HCR-based detection and require further advance to overcome them. With this purpose, here we report a universal and signal-on HCR based detection platform via innovatively coupling CRISPR²⁵ (clustered regularly interspaced short palindromic repeats)-Cas12a system with the enzyme-free signal amplification circuits. The as-termed CRISPR-HCR detection (Scheme 1) turns the traditional “Off signal” into an “On signal”. Meanwhile, the essential HCR components don''t have to be changed. That says, no matter which target is to be detected, an efficient, non-tailed HCR reaction can be adapted without laborious and challenging rebuilding. We have experimentally demonstrated the high efficiency and universality of CRISPR-HCR strategy through applying it for detection of three different targets just by changing crRNA sequences, which are short segments from African swine fever virus (ASFV) p72 gene (dsDNA-T1) and Human Papilloma Virus (HPV) DNA (dsDNA-T2) as well as a random ssDNA (ssDNA-T), which are 21 bp, 20 bp and 20 nt respectively. By coupling loop-mediated isothermal amplification (LAMP) as an upstream amplifier, the CRISPR-HCR strategy can further achieve a practical sensitivity of ∼aM of ASFV gene in serum. Finally, by successfully replacing HCR with another nucleic acid circuits of general interest, catalytic hairpin assembly (CHA), further demonstrating the flexibility of the platform.Open in a separate windowScheme 1Schematic illustration of the CRISPR-HCR platform. (a) Principle of CRISPR-Cas12a-mediated cis and trans cleavage of DNA. (b and c) CRISPR-HCR platform. Without the target sequence, I₀ activates the HCR assembly and produces low fluorescence background due to FRET (b). After target binds with crRNA, Cas12a cleaves I₀ and remains HCR unreacted, producing increased fluorescence targeting signal (c).The detailed CRISPR-HCR pathway is illustrated in Scheme 1. As well-reported, Cas12a can be programmed with CRISPR RNAs (crRNAs) to specifically recognize the target DNA.²⁶ Its collateral cleavage of the nonspecific single stranded DNA (ssDNA) reporter (trans-cleavage), initiated by the recognition and cleavage of the target DNA (cis-cleavage), has been used for the detection of nucleic acids (Scheme 1a).^27,28 Based on our preliminary experiment, it is proved that ssDNA I₀ can be cleaved by CRISPR Cas12a in Fig. S1.^† In our CRISPR-HCR platform (Scheme 1b and c), H1, H2, initiator I₀ (a 24 nt ssDNA), crRNA and Cas12a are the primitive elements. A fluorophore (FAM) and a quencher is labelled on H1 (FAM-H1) and H2 (H2-BlackQ1) respectively. In the absence of target (Scheme 1b), the I₀ can initial a classic HCR reaction to generate [FAM-H1:H2-BlackQ1]_n polymer. Because of the adjacent position of the fluorophore and quencher, the fluorescence of FAM is quenched, getting low background signal compared with the reaction without I₀. While in presence of the targeting sequence (either ssDNA or dsDNA, Scheme 1c), the Cas12a can recognize the target through the bind of antisense guide RNA (crRNA), forming a specific hairpin scaffold structure. Then the Cas12a undergoes a series of conformational changes in its RuvC active site allowing for cis-cleavage of the target. Trans-cleavage activity is subsequently activated, and indiscriminately cleaves all ssDNA in the surrounding.²⁹ In this way, I₀ is non-specifically cleaved and loses its ability to start the HCR reaction, the FAM and BlackQ1 molecules are still apart far from each other, producing a target dependent high fluorescence intensity compared with the non-target background.As illustrated, the introduction of CRISPR-Cas12a system has successfully turns the HCR reaction from a “signal-off” to “signal-on” response. Also important, due to the non-specific cleavage function of Cas12a to I₀, one set HCR components (I₀, H1, and H2) can be suitable to any targeting sequence without rebuilding. Therefore, here we directly select the sequences from one of the most powerful HCR reaction.²⁴In initial principle verification and optimization, we use a piece of ds-DNA fragment within ASFV p72 (ref. 30) as the model target, being named dsDNA-T1. The sequence of dsDNA-T1 is rationally selected starting from a potential PAM site starting from “TTTA” at 5′ end. Its crRNA guide sequence (crRNA-T1) is designed to include antisense of dsDNA-T1 and a hairpin structure. Fig. 1a experimentally verifies the introduction of CRISPR-Cas12a system indeed turns the HCR from a “signal-off” response (Fig. 1a, left) to a “signal-on” one (Fig. 1a, right).Open in a separate windowFig. 1Performance verification for CRISPR-HCR platform. (a) Signal (fluorescence spectra) comparison between traditional HCR and CRISPR-HCR strategy. (b) 3D-Barograph for the optimization of the signal-to-background ratio of CRISPR-HCR. (c) Barograph of the fluorescence intensity to validate the universality of the CRISPR-HCR with different targets, including dsDNA-T1 (blue), dsDNA-T2 (orange) and ssDNA-T (purple), the real-time fluorescence signal has been shown in Fig. S2.^† The error bars represent standard deviation from two independent tests.The experimental condition used in Fig. 1a is selected from a series of optimization. 300 nM Cas12a is used because its presences the highest signal-to-background ratio (Fig. S3^†). We also investigate two critical factors that may usually affect the HCR module, the concentration ratio of I₀ to H1 and H2 and the concentration of H1 (or H2). For convenience, the final concentrations of H1 and H2 are always equal. According to the 3D-bargraph shown in Fig. 1b, among all the conditions tested, the highest signal-to-background ratio appears when the concentration of H1 (or H2) is 240 nM, and the concentration ratio of I₀ to H1 (or H2) is 1 : 3.Based on the optimized conditions, we evaluate the sensitivity of the CRISPR-HCR for detection of the dsDNA-T1. A broad dynamic range (pM to nM, Fig. S4 and S5^†) of more than three orders of magnitude is achieved, with the lowest recognition limit around 1 pM. Furthermore, the universality of the CRISPR-HCR platform is verified by changing the crRNA guide sequence according to a new targeting sequence. Here another dsDNA fragment from Human Papilloma Virus²⁶ (HPV, named dsDNA-T2) and a randomly designed ssDNA sequence (named ssDNA-T) are selected as the models. As shown in Fig. 1c, under the same condition the responses of all three targets (dsDNA-T1, dsDNA-T2, and ssDNA-T) are very close, presenting equally high signal-to-background ratios.Through above proof-of-concept experiments, we have obtained a universal, flexible, and both dsDNA and ssDNA targets. In order to realize more practical performance which can be generalized to those real-world pathogen genes existing at very trace amounts, we further couple an ultra-isothermal nucleic acid amplification with CRISPR-HCR. LAMP reaction^31,32 is selected as the model because it can amplify 10⁹ to 10¹⁰-fold at a constant temperature (60–65 °C) within 1–2 hours.^33,34 In details shown in Fig. 2a and b, the target in above demonstration (Scheme 1c) is replaced by the LAMP amplicons for a special targeting gene. At first, the ASFV gene including the dsDNA-T1 fragment (named dsDNA-ASFV), which is selected as the model-target, has been selected as our template. The LAMP primers are self-designed. The efficiency of the LAMP reaction is verified using traditional agarose gel electrophoresis (Fig. S6^†). We think that the assay detection limit depended on the LAMP detection limit, based on our experiments, the detection limit of the LAMP reaction is 1.25 copies per μL when the detection rate is 95% (Fig. S7^†). To further evaluate the sensitivity of the LAMP-CRISPR-HCR platform, the LAMP products amplified from different concentrations (0.5 copies per μL to 5000 copies per μL) of synthetic dsDNA-ASFV (named ASFV-LAMP-products) are used to start the downstream CRISPR-HCR platform. The fluorescence spectrum (Fig. 2b) clearly shows ASFV-LAMP-products amplified from as low as 0.5 copies per μL (∼aM) dsDNA-ASFV can already generate very high fluorescence enhancement compared with the background of water control (labelled with “0 copy”). It is notable, according to the Fig. S7,^† the detection rate of 0.5 copies per μL is about 25%, and theoretically more than 5 copies per μL can achieve 100% detection rate. On contrast, the signals (Fig. 2c) for LAMP products amplified from genes of non-specific pathogens, for example, Middle East Respiratory Syndrome³⁵ (named MERS-LAMP-products) and M13mp18 DNA³² (named M13mp18-LAMP-products), are almost same as that of water control (labelled with “NC”), which verifies the practically high sensitivity and specificity of the LAMP-CRISPR-HCR platform.Open in a separate windowFig. 2Coupling LAMP with CRISPR-HCR platform (LAMP-CRISPR-HCR). (a) Schematic illustration of CRISPR-HCR system coupling with LAMP reaction. (b) Fluorescence spectra of CRISPR-HCR for ASFV-LAMP-products amplified from different amounts of synthetic dsDNA-ASFV. (c) Fluorescence spectra of the CRISPR-HCR for LAMP products amplified from specific target (5000 copies per μL dsDNA-ASFV), and non-specific targets (water control “NC”, 20 000 copies per μL synthetic dsDNA-MERS and dsDNA-M13mp18). dsDNA-MERS and dsDNA-M13mp18 is, in respective, the synthetic DNA sequence segmented from MERS-CoV and M13mp18 gene. (d) Fluorescence spectra of CRISPR-HCR for ASFV-LAMP-products amplified from water control “NC” and different dilutions of the dsDNA-ASFV extracts in pig serum sample.To test the feasibility of applying the LAMP-CRISPR-HCR strategy in real samples, we explore the detection of dsDNA-ASFV in pig serum, one of the most challenging matrices that contains many interfering molecules. We prepare the pig serum samples spiked with 1 × 10¹⁴ copies per μL of dsDNA-ASAV templates. After extracting the dsDNA-ASFV by the commercial extraction TIANamp genomic DNA kit, the extracts are diluted by different folds, being followed by LAMP-CRISPR-HCR detection. It is found when the sample is diluted by 10¹³ folds, the dsDNA-ASFV can still be detected with very high signal-to-background ratio (Fig. 2d). This result shows the components in real samples cannot affect the detection efficiency, which verifies the practicability of the platform.In order to further evaluate the adaptability of the method, we replace the hybridization chain reaction (HCR) with catalytic hairpin assembly (CHA), which is another widely-used nucleic acid circuit.^1,3,36 According to Fig. 3a, CHA also requires two hairpin substrates (named H3 and H4-FAM), in which H4 is FAM tagged. In the absence of the target, the CHA catalyst C1 (kind of I₀ for HCR) initiates two strand-displacement (SD) reactions that forms H3:H4-FAM:C1 intermediate. Then C1 automatically dissociate from the intermediate and starts another reaction cycle, leaving H3:H4-FAM as the assembly product. At the same time, the tail of H3:H4-FAM is able to start another SD reaction to bind a probe sequence labelled with BlackQ1 (named P-BlackQ1), finally forming the H3:FAM-H4:P-BlackQ1 complex. Very similar to the CRISPR-HCR, once the targeting sequence exists, the Cas12a can recognize the target through the bind of antisense guide RNA (crRNA). Thus, C1 is non-specifically cleaved, remaining the CHA unreacted. An enhanced fluorescence signal is produced (Fig. 3b). Taking dsDNA-T1 as the model-target, the CRISPR-CHA conditions are optimized (Fig. S8–S10^†). Then as low as 1.25 nM dsDNA-T1 can lead to saturated fluorescence enhancement compared with the negative background (labelled with “NC”), as shown in Fig. 3c. Further, via replacing the dsDNA-T1 with LAMP products, we also couple the LAMP with the CRISPR-CHA strategy taking dsDNA-ASFV as the model target. As expected, Fig. 3d shows the ASFV-LAMP-products from 500 copies of dsDNA-ASFV (labelled with “PC”) has successfully produce much higher signal compared that of negative control (labelled with “NC”).Open in a separate windowFig. 3Verification of the flexibility via coupling CRISPR with CHA circuit (CRISPR-CHA). (a and b) Schematic illustration of CRISPR-CHA. Without the target sequence, C1 activates the CHA assembly and produces low fluorescence back-ground due to FRET (a). After target binds with crRNA, Cas12a cleaves C1 and remains CHA unreacted, producing increased fluorescence targeting signal (b). (c) Fluorescence kinetic curves of CRISPR-CHA without (“NC”) and with different concentrations of dsDNA-T1, in presence of C1, H3, H4-FAM and BlackQ1-P:cP. (d) Barograph of the fluorescence intensity of CRISPR-CHA detection for the ASFV-LAMP-products amplified from water control (“LAMP-CRSIPR-CHA NC”) and 500 copies per μL dsDNA-ASFV (“LAMP-CRSIPR-CHA PC”). The error bars represent standard deviation from two independent tests.In this communication, a specific, sensitive, and universal detection platform has been established via coupling CRISPR-Cas12a system and nucleic acid signal amplification circuits. The hybridization chain reaction, HCR, has been taken as the main model circuit. With the assistance of CRISPR-Cas12a system, HCR with traditional “off” signal has been improved to an “on” response. And a well-optimized, high-performance circuit can be generalized to any targeting sequence, either ssDNA or dsDNA. By combing an upstream ultra-amplifier (i.e., LAMP), the so-called CRISPR-HCR platform can detect pathogen genes down to aM level, meeting the high requirement of real-world applications. Finally, similar platform is proven also applicable to other circuits via replacing HCR with CHA circuit. The high adaptability is further confirmed.     

Detection of the thermostable direct hemolysin gene (tdh) and the thermostable direct hemolysin-related hemolysin gene (trh) of Vibrio parahaemolyticus by polymerase chain reaction.

Polymerase chain reaction (PCR) protocols were established for specific detection of the tdh and trh genes, the virulence marker genes of Vibrio parahaemolyticus encoding two related hemolysins. The tdh and trh genes are known to have sequence divergence of up to 3.3% and 16%, respectively. Attempts were made to find suitable primer pairs and annealing temperatures to detect each gene without fail. DNAs extracted from 36 representative strains of V. parahaemolyticus were used in the initial screening with various combinations of primer pairs and annealing temperatures. The combinations of primer pairs and annealing temperatures selected were then tested with DNAs extracted from 227 more strains of V. parahaemolyticus and from 133 bacterial strains belonging to 40 species other than V. parahaemolyticus. PCR protocols (primer pairs and annealing temperatures) were established that gave identical results to those obtained with the tdh- and trh-specific polynucleotide probes. These protocols established for the tdh and trh genes could detect 400 fg (100 cells) of cellular DNA carrying the respective gene. Spike experiments demonstrated that the sensitivities of the established PCRs were reduced by a factor of 10(4)-10(5) by an inhibitor(s) present in a normal faecal sample, indicating the need for either DNA extraction or enrichment of the faecal sample in alkaline peptone water for 4 h before the PCR of faecal samples.     

一起副溶血性弧菌引起食物中毒实验室溯源检测

目的 分析一起食物中毒中分离的副溶血性弧菌(Vibrio parahaemolyticus,Vp)的血清群分布、毒力基因携带情况及分子分型特征,为溯源提供依据。 方法 血清凝集试验检测菌株血清型别,多重PCR方法检测Vp三种毒力基因tdh(耐热直接溶血素),trh(相对耐热直接溶血素)与tlh(不耐热溶血素)的携带情况。肠道细菌重复基因间共同序列(enterobacterial repetitive intergenic consensus sequence,ERIC)PCR分型技术分析不同来源菌株基因型特征。 结果 24株分离株中主要血清群为O3:K6(33.3%),均携带tlh基因、均不携带trh基因。66.7%(16/24)的菌株携带tdh基因,O3:K6血清群菌株均携带tdh基因。ERIC PCR分型分为3个克隆群:E1群包括4株O1,2株O2,O4、O5各1株,E2群包括3株O1,2株O5,O2、O4、O10各1株;E3群包括8株O3。 结论 该起食物中毒是由多种型别Vp引起。主要血清群为O3:K6,在食品与患者中都分离出该血清群的菌株,经ERIC分型,这些菌株来自同一个克隆群。除O3血清群外,从食品、加工存放食品的器具及患者中分离出其他不同血清群Vp,经ERIC分型,不同血清群分为2大克隆群,提示此次食物中毒的传染源可能与食品、加工存放食品器具污染Vp相关。     

Report of a wound infection caused by Vibrio parahaemolyticus and Vibrio vulnificus

The present case describes a foot wound caused by a clam shell from which both Vibrio parahaemolyticus and Vibrio vulnificus were recovered. Although extraintestinal infections associated with Vibrio parahaemolyticus have been reported previously, the simultaneous isolation of two marine vibrios from our case suggests that these organisms may coexist in mixed infections from a common source.