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.

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.

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 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。

A droplet digital PCR method(ddPCR)for sensitive detection and quantification of Carassius auratus herpesvirus(CaHV)

Carassius auratus herpesvirus(CaHV)is a pathogen isolated from crucian carp(Carassius auratus)associated with high mortality.A diagnosis method that can detect the virus at an early stage,specifically and accurately,is an urgent requirement for the prevention of CaHV transmission.In the present study,a droplet digital PCR(ddPCR)method based on the tumor necrosis factor receptor(TNFR)gene was established to detect and quantify CaHV DNA with high specificity and no cross-reactions with other aquatic viruses.Skin mucus samples were collected from infected crucian carp from Day 1–8 after infection,and positive amplification was detected on the first day by ddPCR(0.54 copies/μL),whereas the presence of CaHV was not detected by routine PCR until Day 6.Tissue DNA was then collected from the head kidney of 20 fishes which were injected with CaHV and died during the experiment.The five negative samples checked by routine PCR were detected by ddPCR and real-time PCR(qPCR),respectively.The results showed that the positive detection rate of ddPCR(100%)was higher than that of qPCR(40%).The detection limit of the ddPCR was found to be 0.52 copies/μL,which was much lower than the 50.12 copies/μL determined by qPCR.Overall,ddPCR offers a highly promising diagnosis method for the absolute quantification of CaHV in carrier fish and samples from the skin mucus and head kidney with low viral concentrations.

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。

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 genogroupⅡ(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 ddPCR-based 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.

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。

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。

Super-ARMS PCR和ddPCR检测晚期肺腺癌患者血浆循环肿瘤DNA EGFR基因T790M突变的临床价值研究

目的探讨超级扩增阻滞突变系统PCR(Super-ARMS PCR)和微滴式数字PCR(ddPCR)检测晚期肺腺癌患者经表皮生长因子受体抑制剂(EGFR-TKI)治疗后血浆循环肿瘤DNA(ctDNA)表皮生长因子受体(EGFR)基因T790M突变情况和应用价值。方法经EGFR-TKI治疗后耐药的124例患者分别应用Super-ARMS PCR法和ddPCR法检测T790M突变情况,比较2种方法检出率,用药时间及突变丰度的相关性。结果2种方法共检出51例T790M突变,诊断结果一致性较好,Kappa=0.756;2种方法阳性符合率为74.00%,阴性符合率98.65%,总符合率88.71%。用药超过12个月的患者采用Super-ARMS PCR法检测T790M突变的检出率高于用药小于12个月的患者(P<0.05)。ddPCR法检测突变丰度为0.01%~68.10%。结论2种方法在晚期肺腺癌患者经EGFR-TKI治疗后T790M突变检测中具有较高的一致性,ddPCR法灵敏度更高且可以提供突变丰度的信息。

猪流行性腹泻病毒微滴式数字PCR定量检测方法的建立及初步应用

旨在建立一种敏感性高、特异性强、重复性好的猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)微滴式数字PCR(droplet digital PCR,ddPCR)定量检测方法。本研究根据GenBank登录的PEDV(MK862249.1)M基因序列保守区设计合成特异性引物对和探针;通过对反应体系和反应条件的优化,成功建立了可用于检测PEDV的荧光定量PCR(fluorescent quantitative real-time PCR,FQ-PCR)方法,将自行建立的FQ-PCR方法的引物对/探针用来确定ddPCR方法;对ddPCR方法进行敏感性、特异性、重复性和初步应用试验。结果显示:自行建立的FQ-PCR方法在敏感性、重复性等方面均优于行标FQ-PCR方法;根据自行设计的FQ-PCR方法建立的ddPCR方法最低检测极限为0.15 copies·μL^(-1),敏感性高于行业标准(SN/T 1699—2017)FQ-PCR方法(1.0×10^(1)copies·μL^(-1));模板浓度为1.0×10^(0)~1.0×10^(4)copies·μL^(-1)时,具有良好的线性关系(R 2>0.99);批内、批间变异系数(CV%)为1.52%~7.40%;对猪丁型冠状病毒、猪伪狂犬病毒等11种对照病毒核酸检测结果均为阴性;分别用建立的ddPCR方法、行业标准FQ-PCR方法对150份临床样品进行PEDV核酸检测,ddPCR方法对行业标准FQ-PCR方法检测阳性样品的检测符合率为10^(0)%。本研究成功建立的PEDV ddPCR检测方法可用于临床上PEDV感染的早期检测和定量检测,为PEDV核酸标准物质的研制提供了定量手段。

血小板HPA-3,HPA-15基因分型微滴式数字PCR检测体系的构建

目的建立血小板HPA-3,HPA-15基因分型的微滴式数字PCR(ddPCR)高灵敏检测方法,并初步探索应用于孕妇外周血胎儿游离DNA HPA抗原相容性检测的可行性。方法针对HPA-3,HPA-15的SNP突变位点,设计特异性引物及MGB探针,优化ddPCR退火温度及引物浓度等扩增条件,建立最佳反应体系,明确检验程序。对该检测方法进行方法学性能评估包括特异性、灵敏度、重复性和稳定性。利用ddPCR技术对2022年6月至2023年6月67例临床血液标本进行检测,将等位基因分型结果与基因测序结果比较,并对52例母体外周血胎儿游离DNA HPA抗原进行检测。结果检测血小板HPA-3,HPA-15的ddPCR方法,引物及探针特异性良好,HPA-3,HPA-15的最佳退火温度分别为:61.6℃,60.2℃;体系最佳引物浓度分别为:900 nM,700 nM;探针终浓度均为250 nM。拷贝数定量检测范围为:2~20000 copies,检测下限为0.1 copies/μL且线性良好。在低拷贝数标本中,HPA-3及HPA-15实际检测值的批内及批间变异系数(CV)均<5%。对67份人血液标本DNA的HPA-3,HPA-15基因型检测,结果与基因测序结果完全一致。应用于胎母血小板HPA-3,HPA-15基因型检测结果符合预期。结论本研究构建的HPA-3,HPA-15 ddPCR检测体系准确性高,重复性及稳定性较好,灵敏度高,可应用于临床血小板HPA-3,HPA-15基因型供者库的建立、基因配型及胎母血小板相容性检测等。

BEAMing ddPCR与Super ARMS检测EGFR酪氨酸激酶抑制剂治疗后非小细胞肺癌患者循环肿瘤DNA EGFR基因突变的比较研究

背景与目的:BEAMing微滴数字PCR(droplet digital PCR,ddPCR)被认为是灵敏度极高的基因突变检测方法,探讨该方法和扩增阻碍突变系统(Super amplification refractory mutation system,Super ARMS)检测表皮生长因子受体(epidermal growth factor receptor,EGFR)酪氨酸激酶抑制剂(tyrosine kinase inhibitor,TKI)治疗非小细胞肺癌(non-small cell lung cancer,NSCLC)患者后循环肿瘤DNA(circulating tumor DNA,ctDNA)EGFR基因突变的灵敏度差异,进而为指导临床ctDNA检测方法的选择提供更多参考。方法:收集复旦大学附属肿瘤医院2017-2018年接受过EGFR TKI治疗的NSCLC患者血浆33例及国家病理质控评价中心(PathologyQualityControlCenter,PQCC)模拟血浆10例,采用凯杰血浆游离核酸纯化试剂盒进行循环游离DNA(circulating free DNA,cfDNA)提取,分别用ddPCR和Super ARMS方法进行EGFR基因19 del、T790M和L858R突变检测,两种结果进行对比和统计学分析。结果:33例临床样本应用两种方法检测19 del、T790M和L858R突变,阳性率分别为27.3%vs 27.3%(P=1.00)、42.4%vs 27.3%(P=0.23)和27.3%vs 27.3%(P=1.00)。10例PQCC样本结果与标准结果相比,ddPCR检测T790M符合率显著高于Super ARMS(P=0.01)。ddPCR和Super ARMS检测T790M的样本突变丰度范围分别为0.04%~7.66%和0.05%~7.66%。11例T790MddPCR(+)/Super ARMS(-)的平均突变丰度为0.19%(0.04%~0.76%),与10例Super ARMS(+)平均突变丰度[1.73%(0.05%~7.66%)]差异有统计学意义(P=0.03)。EGFR TKI耐药突变T790M突变丰度在0.01%~0.20%、0.20%~1.00%和>1.00%区间的分布分别为42.9%、14.2%和42.9%。结论:BEAMing ddPCR法较Super ARMS法具有更高的灵敏度,可检出更多T790M耐药患者,可能为更多患者选择最有效的靶向治疗方案提供参考。

基于ddPCR检测荷斯坦牛自由马丁现象的研究

为了鉴定异性双胎牛性器官发育紊乱疾病——牛自由马丁(freemartinism)综合征,试验选择7头中国荷斯坦异性孪生双胎的母牛进行微滴数字PCR(droplet digital PCR,ddPCR)检测,并根据异性孪生母犊不孕的个体染色体为XX/XY嵌合、正常母牛染色体为XX来判定是否为嵌合体。结果表明:此7个样本均存在XX/XY染色体嵌合,均为异性孪生不孕母犊。说明ddPCR方法用于鉴定牛自由马丁现象是有效的。

水貂阿留申病毒的微滴式数字PCR方法的建立及初步应用

为建立水貂阿留申病毒(AMDV)微滴式数字PCR (ddPCR)检测方法,本研究根据AMDV VP2基因的保守区设计特异性引物和探针,通过PCR扩增AMDV VP2基因并克隆至p MD19-T载体,构建重组质粒p MD19-T-AMDV,并经PCR和测序鉴定后利用该质粒标准品作为模板,通过各反应条件的优化,初步建立了检测AMDV的ddPCR方法。分别以AMDV、水貂肠炎细小病毒、犬瘟热病毒、犬细小病毒、水貂流感病毒基因组为模板,按照本研究建立的ddPCR方法检测,评估其特异性;将1.43×10^(7)拷贝/μL~1.43×10^(0)拷贝/μL的重组质粒标准品作为模板,采用优化后的ddPCR和文献中的荧光定量PCR (qPCR)检测,评估该方法的敏感性;分别以不同浓度的重组质粒标准品作为模板,采用优化后的ddPCR分别进行批内和批间重复性试验,以变异系数(CV)评估该方法的重复性。优化结果显示,该方法的最佳引物终浓度为400 nmol/L (1.2μL)、探针终浓度为200 nmol/L (0.6μL)、退火温度为60℃。特异性试验结果显示,该方法仅可检测到AMDV和质粒标准品,而其他相关病毒均为阴性结果。敏感性试验结果显示,该方法对重组质粒标准品的检测限为1.43拷贝/μL,qPCR对质粒标准品的检测限为1.43×10^(2)拷贝/μL,前者比后者的敏感性高100倍。重复性试验结果,批内和批间重复性试验的变异系数均小于3%。对采集的70份病貂肝脏、脾、肾脏、环境拭子、肛拭子样品进行ddPCR和qPCR检测,结果显示,ddPCR对AMDV的检出率为24.28%(17/70);qPCR的检出率为15.7%(11/70),二者的阳性符合率为64.7%,阴性符合率为89.83%,总符合率为91.42%。上述结果表明,本研究建立的ddPCR方法特异性强、敏感性高和重复性好,可以检测各种临床样品及含量极低样品中的AMDV,为AMDV早期感染的检测、流行病学调查及AMD的防控提供有力技术手段。