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.

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.

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

AIM To assess KRAS G12 D mutation detection by droplet digital PCR(dd PCR) in stool-derived DNA from colorectal cancer(CRC) patients.METHODS In 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 paraffinembedded(FFPE) tumor tissues. The KRAS G12 D mutation was then analyzed by dd PCR 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 dd PCR was performed for these patients as well.RESULTS Among 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 G12 D(14.29%, n = 10), which was more frequent in early-stage tumors(I-IIA, n = 7). In agreement with pyrosequencing results, the KRAS G12 D mutation was detected by dd PCR in FFPE tumor-derived DNA, and only a residual number of mutated copies was found in WT controls. The KRAS G12 D mutation was also detected in stool-derived DNA in 80% of all fecal samples from CRC patients with this point mutation. CONCLUSION dd PCR is a reliable and sensitive method to analyze KRAS G12 D 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.

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。

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。

Drop-off微滴式数字PCR检测急性髓系白血病NRAS基因突变及其临床应用

目的:建立检测急性髓系白血病(acute myeloid leukemia,AML)神经母细胞瘤RAS病毒癌基因同源物(NRAS)基因突变的drop-off微滴式数字PCR(droplet digital PCR,ddPCR)方法,并评价该方法的临床应用价值。方法:针对NRAS G12和G13突变位点设计特异性drop-off ddPCR引物和探针,建立最佳反应体系,并对该方法进行性能验证。应用该方法对140例临床样本进行初筛,其检测结果与Sanger测序进行比较,并用二代测序(next generation sequencing,NGS)进行验证。结果:建立drop-off ddPCR检测NRAS基因G12/G13突变的方法,灵敏度达0.158%,重复性、线性良好,其空白限为5.40拷贝数/μL,最低检测下限为15.84拷贝数/μL。在140例初诊AML患者中,drop-off ddPCR检出NRAS突变28例(20%),其突变负荷为0.26%~52.85%(中位2.14%);而Sanger测序仅检出7例(5%)阳性,为进一步验证,将21例Sanger测序结果阴性而drop-off ddPCR检测阳性的样本进行NRAS基因NGS,检出14例阳性,而另外7例NGS阴性的样本,其drop-off ddPCR检测突变频率为0.53%~1.50%(中位1.10%)。结论:建立了drop-off ddPCR技术检测AML患者中NRAS基因突变的方法,该方法灵敏度高,可用于对NRAS基因G12/G13突变进行定量检测,有望可用于AML患者预后判断和疾病监测。

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。

野生型非洲猪瘟病毒多重数字PCR方法的建立

为建立一种鉴别诊断野生株和基因缺失株非洲猪瘟病毒(African swine fever virus,ASFV)的方法,针对ASFV的B646L、EP402R、DP96R基因保守序列设计3套引物和探针,优化三重荧光定量PCR体系作为基础建立微滴式数字PCR(droplet digital PCR,dd PCR)检测方法,并对方法的特异性、灵敏性及重复性进行评估。结果显示,优化后的多重荧光定量PCR检测方法的标准曲线线性关系良好,对PRRSV、PEDV、PRV、VSV、PCV、RNase-free水、重组质粒标准品进行特异性检测,特异性良好;重复性试验结果显示,变异系数均小于2%;对3种重组质粒的最低检测下限均为102 copies/μL。参照上述多重荧光定量PCR方法优化后的条件,进行多重ddPCR检测方法的建立,并且对该方法进行评价。结果显示,ddPCR检测方法标准曲线的线性关系良好;特异性及重复性良好;最低检测下限B646L为11.6 copies/μL、EP402R为13.3 copies/μL、DP96R为16.9 copies/μL;ddPCR的检测灵敏度比荧光定量PCR有优势。使用建立的多重ddPCR方法对14份P3实验室中的样品进行实验室样品检测,样品检测符合率达到100%。成功建立了能够同时检测用于鉴别野毒株感染与疫苗株的荧光定量PCR和微滴式ddPCR方法。

小反刍兽疫病毒液滴式数字PCR检测方法的建立及应用

为实现小反刍兽疫病毒(Peste des petits ruminants virus,PPRV)的快速、准确、定量检测,以国内PPRV Clone 9疫苗株N基因为靶位点设计特异性引物及探针,建立了液滴式数字PCR(Droplet digital PCR,ddPCR)方法,并对其特异性、敏感性及重复性进行评价。将所建立的ddPCR方法与实时荧光定量PCR(Fluorescence quantitative PCR,qPCR)方法的灵敏度进行比较分析并应用于PPRV(Clone 9株)核糖核酸标准物质的定量。结果显示,所建立的ddPCR方法特异性好,仅PPRV检测结果为阳性,其他羊源病毒类生物制品及毒种检测结果均为阴性;敏感性高,基因拷贝数检出限度为4.33拷贝/μL,比qPCR方法的灵敏度(57.3拷贝/μL)高10倍;重复性好,重复性试验的变异系数为1.8%;定量准确,具有一定资质的9家单位各组测量数据组间变异系数小于5%。试验建立的ddPCR方法能够有效地对PPRV核酸进行快速检测,为PPRV的诊断及绝对定量提供了有效方法。

产气荚膜梭菌α-毒素基因数字PCR方法的建立及其在标准物质研制方面的应用

产气荚膜梭菌(Clostridium perfringens)是一种重要的人兽共患病原菌,α-毒素是其分子生物学检测的主要靶标。试验选取Clper引物及探针建立微滴式数字PCR(droplet digtial PCR,ddPCR)方法,最佳引物浓度0.85μmol/L,探针浓度为0.3μmol/L,退火温度为60℃。利用建立的ddPCR方法检测单核细胞增生李斯特菌、沙门氏菌、大肠杆菌、肠球菌、金黄色葡萄球菌、弯曲杆菌的DNA,结果均为阴性,表明该方法具有较好的特异性。合成产气荚膜梭菌α-毒素基因,制成DNA标准品,选取3个稀释梯度的标准品进行重复试验,组内和组间变异系数均小于5%,证明该方法具有较好的重复性。采用7个稀释梯度的标准品进行重复检测,计算出该方法的检测限为12.39 copies/μL,定量限为33.97 copies/μL。通过对68份临床样品进行检测,证实该方法可用于临床,且用于检测质粒DNA时无明显的基质效应。应用ddPCR方法对标准品进行均匀性和稳定性检验,并联合9家实验室进行定值,最终获得了国家市场监督管理总局颁发的标准物质证书(GBW(E)091237)。α-毒素基因ddPCR方法的建立和DNA标准物的研制,为产气荚膜梭菌分子生物学检测试剂的标准化奠定了基础。

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。

SARS-CoV-2 Omicron变异株多重微滴式数字PCR定量方法的建立及应用

【目的】为建立精准、高效的多重微滴式数字PCR(droplet digital PCR,ddPCR)定量分析检测方法,开发可同时鉴别新型冠状病毒(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)ORF1ab基因、N基因、E基因以及Omicron变异株S基因的检测体系,提高病毒性传染疾病的诊断效率及传播风险监测能力。【方法】通过筛选保守基因序列并设计特异性引物与探针,优化反应体系和扩增程序,对该方法的特异性、灵敏度及稳定性进行评价。以临床样本为实验材料,利用建立的ddPCR方法进行检测和验证,确定阳性检出率。【结果】多重ddPCR反应体系中,各对引物探针对目的片段均能有效扩增,SARS-CoV-2 ORF1ab基因、N基因、E基因以及Omicron变异株S基因灵敏度检测下限分别为0.59、0.68、1.44、1.03 copies/μL。在20份临床样本的核酸检测中,共检出16份阳性样本,阳性率达80%(16/20),经荧光定量PCR方法复测符合率一致。【结论】研究建立的多重ddPCR方法特异性强、灵敏度高,可实现对临床样本中微量新冠病毒的精确定量检测。

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。

基于微滴式数字PCR技术检测树莓制品的定量分析方法

为了检验树莓制品中是否掺杂其他成分,建立了微滴式数字聚合酶链式反应(Droplet Digital PCR,ddPCR)技术定量检测树莓成分的检验方法。结果表明,实验建立的树莓掺假模型的检测结果准确,为检测市售树莓制品中是否掺假提供了一种新的、更加精确的定量手段。

微滴数字PCR在重症急性胰腺炎疑似血流感染病原学诊断中的应用

目的探讨微滴数字聚合酶链反应(ddPCR)在重症急性胰腺炎(SAP)合并疑似血流感染(BSI)病原学诊断中的价值。方法选取2022年7—9月某院重症医学科收治的SAP患者,在疑似BSI发作时同步采集静脉血进行ddPCR检测和血培养(BC)及药敏试验(AST),记录两种检测方法的耗时,比较ddPCR与BC的检测结果,计算ddPCR的病原学诊断效能,并探讨ddPCR检测病原菌载量值与感染指标水平的相关性。结果共纳入22例患者,采集52份静脉血标本进行检测,BC阳性17份(32.7%),检出病原体29株;ddPCR阳性41份(78.8%),检测出病原体73株。ddPCR耗时低于BC[(0.16±0.03)d VS(5.92±1.20)d,P<0.001]。在ddPCR检测范围内,以BC为金标准,ddPCR检测的灵敏度和特异度分别为80.0%、28.6%;联合检测前1周内非血标本微生物证据综合判定BSI,ddPCR检测的灵敏度和特异度分别提高至91.9%、76.9%。ddPCR耐药基因检测中,19份检出bla KPC,9份检出bla NDM/IMP,6份检出V an A/V an M,5份检出mec A。相关性分析显示病原菌载量值与C反应蛋白、降钙素原水平呈正相关(r分别为0.347、0.414,均P<0.05)。结论ddPCR作为一种辅助BC诊断BSI的检测方法具有灵敏度高、耗时低等优势,值得进一步探讨其在临床中的应用。

鸡传染性喉气管炎病毒微滴式数字PCR定量检测方法的建立

旨在建立检测鸡传染性喉气管炎病毒(infectious laryngotracheitis virus, ILTV)绝对定量方法。根据已发表的鸡ILTV的TK基因序列,针对其保守区域分别设计1对特异引物和1条探针,建立检测鸡ILTV的微滴式数字PCR(droplet digital PCR,ddPCR)方法,并评价其特异性、敏感性和重复性。结果显示,建立方法的最佳引物浓度为20μmol/μL,最佳探针浓度为10μmol/μL,最佳退火温度为56℃。特异性检测结果显示,建立的方法只检出ILTV,没有检出其他病原株。敏感性检测结果显示,采用建立的方法定量检出ILTV重组质粒标准品的最低限为4.6拷贝/μL。对3个连续稀释的pMD18-ILTV重组质粒DNA进行检测,3次重复检测结果的变异系数均小于5%。对83份病鸡喉拭子、肺及脾组织样品进行检测,采用建立的ddPCR检出ILTV阳性样品10份,荧光定量PCR检出ILTV阳性样品9份,ddPCR的阳性检出率(12.05%)高于荧光定量PCR的阳性检出率(10.84%)。结果表明,建立的ddPCR方法定量检测ILTV特异性强、敏感性高、重复性好,为绝对定量检测ILTV提供更好的技术支撑。

狂犬病病毒微滴式数字PCR检测方法的建立

为了建立可绝对定量狂犬病病毒(RABV)的微滴式数字PCR(ddPCR)检测方法,本试验根据RABV基因组起始区域设计引物和探针,继而优化引物和探针浓度以及退火温度,并对建立的ddPCR方法的灵敏度、特异性和重复性进行分析。结果显示:ddPCR方法的最佳引物和探针浓度分别为900 nmol/L和250 nmol/L,最佳退火温度为55℃;ddPCR方法的标准曲线相关系数(R~2)为0.999,呈现良好的线性关系;灵敏度高,最低可检测到3.37 copies/μL;特异性良好,与常见犬病原无交叉反应;重复性好,检测结果稳定。临床样品的检测结果显示,本试验建立的狂犬病病毒ddPCR方法与实时荧光定量反转录PCR(RT-PCR)方法的符合率为100%。结果表明,本试验建立的狂犬病病毒微滴式数字PCR方法灵敏度高、特异性强,可用于狂犬病病毒的绝对定量检测。