Protocol for detection of pathogenic enteric RNA viruses by regular monitoring of environmental samples from wastewater treatment plants using droplet digital PCR

Background:The present comprehensive protocol is focused on the detection of pathogenic enteric RNA viruses,explicitly focusing on norovirus genogroup II(GII),astrovirus,rotavirus,Aichi virus,sapovirus,hepatitis A and E viruses in wastewater treatment plants through droplet digital PCR(ddPCR).Enteric viruses are of significant public health concern,as they are the leading cause of diseases like gastroenteritis.Regular monitoring of environmental samples,particularly from wastewater treatment plants,is crucial for early detection and control of these viruses.This research aims to improve the understanding of the prevalence and dynamics of enteric viruses in urban India and will serve as a model for similar studies in other regions.Our protocol's objective is to establish a novel ddPCRbased methodology for the detection and molecular characterization of enteric viruses present in wastewater samples sourced from Bhopal,India.Our assay is capable of accurately quantifying virus concentrations without standard curves,minimizing extensive optimization,and enhancing sensitivity and precision,especially for low-abundance targets.Methods:The study involves fortnightly collecting and analyzing samples from nine wastewater treatment plants over two years,ensuring comprehensive coverage and consistent data.Our study innovatively applies ddPCR to simultaneously detect and quantify enteric viruses in wastewater,a more advanced technique.Additionally,we will employ next-generation sequencing for detailed viral genome identification in samples tested positive for pathogenic viruses.Conclusion:This study will aid in understanding these viruses’genetic diversity and mutation rates,which is crucial for developing tailored intervention strategies.The findings will be instrumental in shaping public health responses and improving epidemiological surveillance,especially in localities heaving sewage networks.

炭疽芽孢杆菌微滴式数字PCR定量检测方法的建立

目的建立炭疽芽孢杆菌的微滴式数字聚合酶链式反应(Droplet digital PCR,ddPCR)方法对炭疽芽孢杆菌实验室活动污染的定量评估提供技术支持。方法以炭疽芽孢杆菌pXO1质粒编码保护性抗原pagA基因为靶序列,优化微滴数字PCR方法的反应条件,建立实验室微环境中炭疽芽孢杆菌核酸定量方法;对比微滴式数字PCR方法和平板计数法的定量评估效果,分析ddPCR的灵敏性、特异性和重复性。结果建立的ddPCR方法最佳引物和探针终浓度分别为900nmol·L^(-1)和250nmol·L^(-1),最佳退火温度为60℃,最佳升降温速度为1℃/s,本方法的最低检测下限为1.12copies·μL^(-1),未发现与常见疫病存在交叉反应,重复性试验的变异系数小于5%。结论本研究中建立的炭疽芽孢杆菌的微滴数字PCR方法敏感性高、特异性强、重复性好,为疫情监测、流行病学调查和实验室污染微环境检测提供重要技术。

Utility of Droplet Digital PCR Assay for Quantitative Detection of Norovirus in Shellfish, from Production to Consumption in Guangxi, China

Objective Shellfish are recognized as important vehicles of norovirus-associated gastroenteritis. The present study aimed to monitor norovirus contamination in oysters along the farm-to-fork continuum in Guangxi, a major oyster production area in Southwestern China. Methods Oyster samples were collected monthly from farms, markets, and restaurants, from January to December 2016. Norovirus was detected and quantified by one-step reverse transcription-droplet digital polymerase chain reaction（RT-ddPCR）. Results A total of 480 oyster samples were collected and tested for norovirus genogroups I and II. Norovirus was detected in 20.7% of samples, with genogroup II predominating. No significant difference was observed in norovirus prevalence among different sampling sites. The norovirus levels varied widely, with a geometric mean of 19,300 copies/g in digestive glands. Both norovirus prevalence and viral loads showed obvious seasonality, with a strong winter bias. Conclusion This study provides a systematic analysis of norovirus contamination ‘from the farm to the fork＇ in Guangxi. RT-ddPCR can be a useful tool for detection and quantification of low amounts of norovirus in the presence of inhibitors found particularly in foodstuffs. This approach will contribute to the development of strategies for controlling and reducing the risk of human illness resulting from shellfish consumption.

Application of droplet digital PCR in detection of seed-transmitted pathogen Acidovorax citrulli

Bacterial fruit blotch caused by Acidovorax citrulli is a serious threat to cucurbit industry worldwide.The pathogen is seedtransmitted,so seed detection to prevent distribution of contaminated seed is crucial in disease management.In this study,we adapted a quantitative real-time PCR(qPCR)assay to droplet digital PCR(ddPCR)format for A.citrulli detection by optimizing reaction conditions.The performance of ddPCR in detecting A.citrulli pure culture,DNA,infested watermelon/melon seed and commercial seed samples were compared with multiplex PCR,qPCR,and dilution plating method.The lowest concentrations detected(LCD)by ddPCR reached up to 2 fg DNA,and 102 CFU mL–1 bacterial cells,which were ten times more sensitive than those of the qPCR.When testing artificially infested watermelon and melon seed,0.1%infestation level was detectable using ddPCR and dilution plating method.The 26 positive samples were identified in 201 commercial seed samples through ddPCR,which was the highest positive number among all the methods.High detection sensitivity achieved by ddPCR demonstrated a promising technique for improving seed-transmitted pathogen detection threshold in the future.

Development of a sensitive and reliable droplet digital PCR assay for the detection of ‘Candidatus Liberibacter asiaticus'

Citrus Huanglongbing（HLB, yellow shoot disease） is one of the most serious citrus diseases worldwide. To better improve the detection sensitivity, a droplet digital PCR（ddPCR） assay was developed for the rapid detection of ‘Candidatus Liberibacter asiaticus＇（Las）, the putative causal agent of HLB. The detection of sensitivity comparison using positive plasmid indicated that dd PCR was superior to quantitative PCR（qPCR） for detecting and quantifying Las at low concentrations. The Las detection of 40 field samples also showed that six of 13 asymptomatic samples（46.15%） with high Ct value（〉35） were positive by dd PCR. This methodology showed great potential for early HLB infection diagnosis.

Application of Digital PCR in the Analysis of Transgenic Soybean Plants

Detection and quantification of transgenes are important in analyzing genetically modified organisms (GMOs). Quantitative polymerase chain reaction (qPCR) is commonly utilized for such purposes. However, qPCR has certain limitations in detecting and quantifying transgenes in GMOs, such as the need of certified reference materials, a standard curve, and possible affection by inhibitors. Therefore, alternative and possibly better methods are needed. Recent advances in digital PCR technologies have promised to allow accurate quantification of nucleic acids and therefore provided another useful technique to analyze GMOs. Thermo Fisher ScientificTM has recently commercialized the Applied BiosystemsTM QuantStudioTM 3D digital PCR system that can be used for a wide range of applications involving nucleic acids. It will be beneficial to the scientific community to show the applicability of this digital PCR system in detecting and quantifying transgenes in GMOs. In the present study, the transgenes present in the Roundup Ready Soybean (RR1, event 40-3-2) and Roundup Ready Soybean 2 (RR2, event MON89788) developed by Monsanto Corporation were analyzed by using this digital PCR system. The qPCR analysis results were included for comparison. Using specifically designed TaqMan assays, as low as 1% of the RR1 or RR2 soybean material was reliably detected and quantified on the dPCR platform. Therefore, digital PCR is a sensitive and reliable method to analyze the RR transgenic soybeans, and should be another useful tool for analyzing other transgenic plants.

Detection of KRAS G12D in colorectal cancer stool by droplet digital PCR

AIMTo assess KRAS G12D mutation detection by droplet digital PCR (ddPCR) in stool-derived DNA from colorectal cancer (CRC) patients.METHODSIn this study, tumor tissue and stool samples were collected from 70 patients with stage I-IV CRC diagnosed by preoperative biopsy. KRAS mutational status was determined by pyrosequencing analysis of DNA obtained from formalin-fixed paraffin-embedded (FFPE) tumor tissues. The KRAS G12D mutation was then analyzed by ddPCR in FFPE tumors and stool-derived DNA from patients with this point mutation. Wild-type (WT) tumors, as determined by pyrosequencing, were included as controls; analysis of FFPE tissue and stool-derived DNA by ddPCR was performed for these patients as well.RESULTSAmong the total 70 patients included, KRAS mutations were detected by pyrosequencing in 32 (45.71%), whereas 38 (54.29%) had WT tumors. The frequency of KRAS mutations was higher in left-sided tumors (11 located in the right colon, 15 in the left, and 6 in the rectum). The predominant point mutation was KRAS G12D (14.29%, n = 10), which was more frequent in early-stage tumors (I-IIA, n = 7). In agreement with pyrosequencing results, the KRAS G12D mutation was detected by ddPCR in FFPE tumor-derived DNA, and only a residual number of mutated copies was found in WT controls. The KRAS G12D mutation was also detected in stool-derived DNA in 80% of all fecal samples from CRC patients with this point mutation.CONCLUSIONddPCR is a reliable and sensitive method to analyze KRAS G12D mutation in stool-derived DNA from CRC patients, especially at early stages. This non-invasive approach is potentially applicable to other relevant biomarkers for CRC management.

A Direct Droplet Digital PCR Method for <i>E. coli</i>Host Residual DNA Quantification

Injectable drugs manufactured in E. coli must be tested for host residual DNA (hr DNA) impurity in ensuring drug purity and safety. Because of low allowable hr DNA as impurity, highly sensitive methods are needed. Droplet digital PCR (ddPCR) is a new method where the reaction is partitioned into about 20,000 nanoliter-sized droplets and each droplet acts as individual PCR reaction. After completion of end-point PCR, droplets are analyzed for fluorescence and categorized as positive or negative and DNA quantified using Poisson statistics. Here we describe development of a direct E. coli hr DNA dd PCR method where the drug is directly added to the ddPCR reaction. We show that the ddPCR method has acceptable precision and high accuracy, works with different biologic drugs, and compared to qPCR shows higher tolerance of drug matrices. The method does not require DNA extraction or standard curves for quantification of hr DNA in unknown samples.

发酵乳中活性益生菌三重数字PCR定量检测方法的建立

益生菌发酵乳是益生菌市场发展的重要赛道,精确定量产品中活性益生菌对保障其营养功能至关重要。文章以嗜酸乳杆菌(Lactobacillus acidophilus)、鼠李糖乳杆菌(Lactobacillus rhamnosus)和动物双歧杆菌(Bifidobacterium animalis)为研究对象,采用种特异性引物和探针,结合叠氮溴化丙锭(propidium monoazide,PMA)染色,基于芯片式数字PCR技术建立了发酵乳中活性益生菌的三重定量检测方法。结果表明,3组引物和探针均能特异性扩增目标菌株,无交叉反应,且20μg/mL的PMA能够有效抑制嗜酸乳杆菌、鼠李糖乳杆菌和动物双歧杆菌死菌DNA的扩增。该检测方法对嗜酸乳杆菌、鼠李糖乳杆菌和动物双歧杆菌的灵敏度分别为1.65、2.20、2.16 copies/μL,重复性检测的相对标准偏差为1.52%~8.69%,成功实现了对发酵乳中3种常见益生菌的活菌定量检测。为益生菌发酵乳中活菌检测的标准化提供了技术支持和科学依据。

Copy number and zygosity determination of transgenic rapeseed by droplet digital PCR

Establishing an accurate and rapid method for copy number and zygosity determination can accelerate genetic engineering research process. In this study, droplet digital PCR (ddPCR), an emerging DNA absolute quantification technology, was used to identify single-copy homozygous transgenic lines from a batch of T0 transgenic rapeseed harboring 11 exogenous elements. Copy number of exogenous gene was evaluated in T0 generation based on calculated ratio between transgene and reference CruA gene, single-copy transformants were selected for selfing followed by subsequent zygosity analysis. Single-copy homozygous transgenic plants were successfully screened out in T1 generation by ddPCR.Segregation analysis with T2 seedlings verified that identification results of ddPCR were accurate and reliable. This study provides a novel rapid and accurate method for copy number and zygosity determination in transgenic rapeseed which overcomes disadvantages of traditional Southern analysis and recently developed real-time quantitative method.

Establishment and Application of Digital RT-PCR Assay for Detection of Avian Influenza Virus H9 Subtype

A digital RT-PCR method for rapid detection of H9 subtype influenza was established by comparing the two methods of digital RT-PCR and real-time quantitative RT-PCR. The sensitivity, specificity and reproducibility of the two methods for H9 were determined by gradient dilution using the same pair of primers and probes. Both methods were able to detect 104 times diluted H9 pathogens, while digital RT-PCR could detect H9 in single droplets, and its sensitivity was higher than real-time quantitative RT-PCR. At the same time, the specificities of both methods were very strong, with no amplification reactions for H3N2, H4N2, H6N2. The reproducibility of the two methods were also good. Digital RT-PCR has a higher sensitivity than real-time quantitative RT-PCR and could play an important role in the rapid detection of H9 subtype influenza virus.

Establishment and Preliminary Application of One-step Reverse Transcriptase Droplet Digital PCR Assay for Bovine Viral Diarrhea Virus

[Objective]The paper was to establish a one-step reverse transcriptase droplet digital PCR(RT-ddPCR)assay for bovine viral diarrhea virus(BVDV).[Method]Based on one-step real-time quantitative PCR(RT-qPCR)assay,BVDV-specific primers and probes were designed in this study.The reverse transcriptase,annealing temperature,primer and probe concentrations and reaction conditions of RT-ddPCR assay were optimized.Meantime,the specificity,sensitivity and repeatability of RT-ddPCR assay were evaluated.[Result]The optimal reverse transcription system for the established RT-ddPCR assay was as follows:commercial one-step reverse transcriptase droplet digital PCR kit with matching reagents,a final primer concentration of 900 nmol/L,a final probe concentration of 250 nmol/L and an optimal annealing temperature of 57℃.The results were negative when the method was used to detect other common epidemic viruses;the minimum detection limit was 3.2 copies/μL with good repeatability,and the coefficient of variation was less than 5%.RT-ddPCR and RT-qPCR assays were used to test 24 bovine swab samples and the test results showed that the established RT-ddPCR assay was superior to RT-qPCR assay.[Conclusion]The RT-ddPCR assay established in this study has strong specificity,high sensitivity and good repeatability,and is suitable for nucleic acid detection of clinical samples.This study provided a technical support for early detection and quantitative diagnosis of BVDV infection.

PMA结合ddPCR检测食品中金黄色葡萄球菌的研究

研究了将叠氮溴化丙锭(PMA)与微滴式数字PCR(ddPCR)技术相结合,用于金黄色葡萄球菌活菌的检测。结果表明,强烈光照15 min,可以使PMA与死菌DNA共价交联,同时钝化游离的PMA;可以有效抑制金黄色葡萄球菌死菌DNAPCR扩增的PMA终浓度为2.0μg/m L;不抑制活菌DNA扩增的PMA最高浓度是5.0μg/m L。在不同死、活菌比例下,PMA-ddPCR可以定量检测活菌,避免了死菌DNA的干扰,本方法的检出限为10 copy/20μL。利用PMA-ddPCR检测人工污染鸡肉样品,最低可检出102cfu/m L的金黄色葡萄球菌。表明PMA-ddPCR方法的灵敏度高。

抗草甘膦转基因玉米外源基因ddPCR拷贝数分析

【目的】本研究是从前期获得的100余份转基因抗除草剂草甘膦玉米品系中,挑选T抗-1等14个转基因玉米品系进行拷贝数检测。【方法】采用一种新型的拷贝数分析方法——微滴数字PCR技术(droplet digital PCR,简称dd PCR),设计特异引物对外源抗草甘膦基因2m G2-epsps进行绝对定量分析。【结果】供试的14份转基因材料中10份是单拷贝品系。同时,通过引物探针特异性试验、叶片基因组DNA的PCR抑制和浓度检测、转基因玉米的外源基因2m G2-epsps的实时PCR检测和基因组DNA的酶切等系列研究,建立稳定的转基因玉米拷贝数分析的dd PCR检测体系。【结论】微滴数字PCR技术为这批转基因玉米的下一步转基因生物安全评价提供了重要参数,同时建立了转基因玉米(genetically modified maize)外源基因拷贝数dd PCR分析方法。

尼罗罗非鱼无乳链球菌微滴式数字PCR检测方法的建立及临床应用

通过设计筛选引物和探针、优化反应浓度和退火温度,构建一种尼罗罗非鱼(Oreochromis niloticus)无乳链球菌(Streptococcus agalactiae)的微滴式数字PCR(dd PCR)检测方法,分析该方法的敏感性、特异性及重复性,并应用于临床样品检测。结果显示:当引物、探针浓度分别为0.9μmol·L^(-1)、0.3μmol·L^(-1)且退火温度为56.9℃时,建立的罗非鱼无乳链球菌dd PCR方法阴、阳性微滴分布界限明显,平均拷贝数高,有较高扩增反应效率;线性关系线良好(R^(2)=0.997 3),最低检测限为2.56 copies·μL^(-1);与猪链球菌2型、鱼类海豚链球菌和其他5种常见的水生动物疫病病原体无交叉反应;重复变异系数为3.15%;临床样品检测结果与实时荧光PCR方法结果的符合率100%,与细菌分离鉴定方法结果符合率为94.12%。结果表明,建立的罗非鱼无乳链球菌dd PCR检测方法灵敏度高、特异性强、重复性好,可对罗非鱼无乳链球菌感染的临床样品进行定量检测,为尼罗罗非鱼无乳链球菌的研究提供有益参考。

基于ddPCR技术分析EB病毒载量特征及与qPCR技术的比较研究

目的解决临床工作中应用实时荧光定量PCR(qPCR)方法检测EB病毒(EBV)载量与临床诊断不符的问题,探讨微滴式数字PCR(ddPCR)和qPCR方法检测EBV载量的能力并为临床提供可能的解决方案。方法收集510例疑似EBV感染相关疾病患者血浆标本,采用ddPCR和qPCR两种方法测定同一血浆标本的EBV-DNA载量。结果 EBV感染人群中,EBV-DNA载量较其他地区低,载量中位数仅360copies/mL,其中初诊未治鼻咽癌患者中位病毒载量为4 590copies/mL,治疗后鼻咽癌患者中位病毒载量下降为430copies/mL,免疫力低下者中位病毒载量为130copies/mL,而淋巴瘤患者中位病毒载量为840copies/mL;qPCR检测EBV感染以400copies/mL为界值,高于400copies/mL时,ddPCR与qPCR的EBV-DNA测定水平值呈中度相关(r=0.533,P<0.05),低于400copies/mL时,ddPCR与qPCR的EBV-DNA测定水平值呈弱相关(r=0.299 5,P<0.05);以ddPCR为标准,qPCR检测EBV-DNA的灵敏度仅为0.317,以ddPCR检测结果为标准,构建qPCR的受试者工作特征曲线下面积为0.871,此时临界值(qPCR)为10copies/mL,灵敏度为0.824,特异度为0.780。结论采用ddPCR方法或优化qPCR的临界值去检测EBV-DNA载量更能为临床诊断EBV感染提供有利支持。

锦鲤疱疹病毒微滴式数字PCR检测方法的建立及临床应用

【目的】构建一种敏感性高、特异性强、重复性好的锦鲤疱疹病毒(Koi herpesvirus,KHV)微滴式数字PCR(ddPCR)检测方法,为锦鲤疱疹病毒的定量检测和低浓度样品检测提供技术支持。【方法】参照靶位基因锦鲤疱疹病毒TK基因(GenBank登录号:KX609547.1)设计合成引物和探针,通过比对筛选出最佳引物探针,优化反应体系和退火温度,建立与实时荧光PCR方法的线性关系,并分析该方法的敏感性、特异性、重复性,最后应用于临床样品检测。【结果】当引物、探针浓度分别为900、300 nmol/L且退火温度为57℃时,建立的KHVddPCR扩增反应效率最高,阴阳性微滴分布界限最为明显。KHVddPCR敏感性强,检测限低至0.46拷贝/μL,在0~8.1×10^(4)拷贝/μL范围内,与实时荧光PCR检测结果的线性关系较佳(R2=0.9949)。检测变异系数低,批内变异系数为2.70%,批间变异系数为3.02%。与鲤浮肿病毒、鲫造血器官坏死病毒、真鲷虹彩病毒、草鱼出血病毒、罗非鱼湖病毒、十足目虹彩病毒、虾肝肠胞虫(Enterocytozoon hepatopenaei)和诺卡氏菌(Nocardia seriolae)等其他8种常见的水生动物疫病阳性样品无非特异性反应。在88份的临床样品检测中,阳性2份,阳性检出率为2.27%;在8份能力验证样品检测中,5份阳性,与能力验证满意结果一致,与实时荧光PCR方法和套式PCR方法检测结果一致。【结论】建立的锦鲤疱疹病毒ddPCR检测方法敏感性高、特异性强、重复性好,可定量检测锦鲤疱疹病毒DNA,为锦鲤疱疹病毒的研究提供参考。

应用于dPCR的大视场荧光显微检测系统的设计

数字聚合酶链式反应(dPCR)作为一种新的核酸检测技术,在生物医学等领域得到广泛的应用。为了进一步提升现有的dPCR检测系统的检测效率,设计了一套基于大视场荧光显微镜的dPCR检测系统,将照明系统与滤波模组设计于显微镜物空间,结合复眼照明系统实现大面积匀光照明,照明面积可达到32mm×22mm,照明均匀性达到80%以上,提出一种结合神经网络的培养皿计数检测方法,可以有效筛选出培养皿中已经蒸发的培养皿。整个系统从相机曝光到检测结束仅需要几秒钟时间,相比传统的图像拼接式检测方法,其检测效率得到了明显提高。

柑橘黄龙病菌亚洲种微滴数字PCR检测方法的建立

柑橘黄龙病是世界柑橘产业的毁灭性病害,带病苗木、带菌接穗和田间病株是病害的初侵染源,建立一套病原菌超灵敏检测方法对柑橘黄龙病的早期预警及防控具有重要意义。根据柑橘黄龙病亚洲种16S rDNA基因序列设计特异引物,建立基于微滴数字PCR技术的检测方法,并评价该方法的灵敏度和检测准确性。结果表明,研究建立的柑橘黄龙病亚洲种微滴数字PCR检测方法特异性强、灵敏度高,其检测灵敏度是荧光定量PCR的10倍。建立的微滴数字PCR检测技术为柑橘种质资源的早期黄龙病检测提供了一种新方法。

一种高通量dPCR荧光图像自适应增强算法

目的为了改善荧光图像背景光照不均匀和对比度低的问题,提出一种荧光图像自适应亮度校正和低对比度增强算法。方法根据光照成像原理,利用引导滤波提取出荧光图像的光照分量,通过改进的二维Gamma函数动态校正背景光照,利用Top-hat变换分离出校正后的前景和背景,对前景进行自适应直方图均衡化,以实现荧光图像自适应增强的目的。结果对比传统算法,文中算法处理后的图像背景光照均匀,对比度增强效果明显,其中标准差平均提高了9.4倍,平均梯度平均提高了1.2倍,信息熵平均提高了0.2倍。结论文中算法可以改善高通量dPCR荧光图像背景光照不均匀性,提高图像对比度,突出图像中隐藏的细节,对其他荧光图像处理也具有参考价值。