Development of TaqMan-based Real-time PCR Assay for Detecting Transmissible Gastroenteritis Virus and Its Application in Vaccine Evaluation

[Objective] This study aimed to establish a TaqMan-based real-time PCR assay for detecting transmissible gastroenteritis virus （TGEV）. [Method] Primers and a probe were designed according to the conserved sequence of N gene in TGEV genome. After gradient dilution, the recombinant plasmid harboring the N gene was used as a standard for real-time PCR assay to establish the standard curve. [Re- sult] The results showed that the established real-time PCR assay exhibited a good linear relationship within the range of 102-10^10 copies/ul; the correlation coefficient was above 0.99 and the amplification efficiency ranged from 90% to 110%. The de- tection limit of real-time PCR assay for TGEV was 10 copies/μl, suggesting a high sensitivity; there was no cross reaction with other porcine viruses, indicating a good specificity; coefficients of variation within and among batches were lower than 3%, suggesting a good repeatability. The established real-time PCR method could be ap- plied in quantitative analysis and evaluation of the immune efficacy of TGEV vac- cines and detection of TGEV in clinical samples. [Conclusion] The TaqMan-based real-time PCR assay established in this study is highly sensitive and specific, which can provide technical means for the epidemiological survey of TGEV, development of TGEV vaccines and investigation of the pathogenesis of TGE.

Evaluation of a New Real-time PCR Assay for Detection of Mycoplasma Pneumoniae in Clinical Specimens

Objective To establish and evaluate a real-time PCR assay to detect Mycoplasma pneumoniae （M.pneumoniae） in clinical specimens. Methods By analysing the whole pl gene sequence of 60 M.pneurnoniae clinical isolates in Beijing of China, an optimized real-time PCR assay （MpP1） using pl gene conserved region was designed. The specificity and sensitivity of this assay were evaluated and compared with other two reported assays （RepMpl and Mp181） using 40 positive and 100 negative clinical specimens. Results The detection limit of the new assay was 8.1 fg （about 1-3CFU） M.pneumoniae DNA. The sensitivity of MpP1, RepMpl, and Mp181 assays appeared to be 100%, 100%, and 85%, respectively. Conclusion MpP1 assay is suitable for the detection of M.pneumoniae in Chinese clinical specimens.

Development and validation of a TaqMan^(TM) fluorescent quantitative real-time PCR assay for the rapid detection of Edwardsiella tarda

Edwardsiella tarda is one of the most important emerging pathogens in tile global aquaculture industries. As such, an accurate diagnosis and quantitative analytical methods are urgently needed for this bacterium. In this study, primers and a TaqMan probe specific to the conservative sequences of the 16S rRNA gene of E. tarda were designed. The concentration of primers and TaqMan probe were optimized to 200 nmol/L and 120 nmol/L, respectively. The detection sensitivity of the FQ- PCR assay was determined to be as low as five copies of the target sequence per reaction using the pGEM-16S rDNA recombinant plasmid as a template, which was 100 times more sensitive than conventional PCR. A standard curve by plotting the threshold cycle values （y） against the common logarithmic copies （logl0n~ as x; n~ is copy number） of pGEM-16S rDNA was generated. The results of intra- and inter-assay variability tests demonstrate that the established FQ-PCR method was highly reproducible. The assay was specific for E. tarda as it showed that there was no cross-reactivity to eight additional bacterial pathogen strains in aquaculture. Thus, the FQ-PCR assay has the potential for diagnostic purposes and for other applications, especially for the rapid detection and quantification of low-grade E. tarda infections.

Development of a real-time PCR assay(SYBR Green I) for rapid identification and quantification of scyphomedusae Aurelia sp.1 planulae

The complicated life cycle ofAurelia spp., comprising benthic asexually-reproducing polyps and sexually-reproducing medusae, makes it hard for researchers to identify and track them, especially for early stage individuals, such as planulae. To solve this problem, we developed a real-time PCR assay （SYBR Green I） to identify planulae in both cultured and natural seawater samples. Species-specific primers targeting Aurelia sp.1 mitochondrial 16S rDNA （mr 16S rDNA） regions were designed. Using a calibration curve constructed with plasmids containing the Aurelia sp. 1 mt 16S rDNA fragment and a standard curve for planulae, the absolute number of mt 16S rDNA copies per planula was determined and from that the total number ofplanulae per sample was estimated. For the field samples, a 100-fold dilution of the sample DNA combined with a final concentration of 0.2 μg/μL BSA in the PCR reaction mixture was used to remove real- time PCR inhibitors. Samples collected in Jiaozhou Bay from July to September 2012 were subsequently analyzed using this assay. Peak Aurelia sp.1 planula abundance occurred in July 2012 at stations near Hongdao Island and Qingdao offshore; abundances were very low in August and September. The real-time PCR assay （SYBR Green I） developed here negates the need for traditional microscopic identification, which is laborious and time-consuming, and can detect and quantify jellyfish planulae in field plankton samples rapidly and specifically.

A multiplex real-time PCR assay for simultaneous detection of classical swine fever virus,African swine fever virus,and atypical porcine pestivirus

With the implementation of the C-strain vaccine,classical swine fever(CSF) has been under control in China,which is currently in a chronic atypical epidemic situation.African swine fever(ASF) emerged in China in 2018 and spread quickly across the country.It is presently occurring sporadically due to the lack of commercial vaccines and farmers’ increased awareness of biosafety.Atypical porcine pestivirus(APPV) was first detected in Guangdong Province,China,in 2016,which mainly harms piglets and has a local epidemic situation in southern China.These three diseases have similar clinical symptoms in pig herds,which cause considerable losses to the pig industry.They are difficult to be distinguished only by clinical diagnosis.Therefore,developing an early and accurate simultaneous detection and differential diagnosis of the diseases induced by these viruses is essential.In this study,three pairs of specific primers and Taq-man probes were designed from highly conserved genomic regions of CSFV(5’ UTR),African swine fever virus(ASFV)(B646L),and APPV(5’ UTR),followed by the optimization of reaction conditions to establish a multiplex real-time PCR detection assay.The results showed that the method did not cross-react with other swine pathogens(porcine circovirus type 2(PCV2),porcine reproductive and respiratory syndrome virus(PRRSV),foot-and-mouth disease virus(FMDV),pseudorabies virus(PRV),porcine parvovirus(PPV),and bovine viral diarrhea virus BVDV).The sensitivity results showed that CSFV,ASFV,and APPV could be detected as low as 1 copy μL–1;the repeatability results showed that the intra-assay and interassay coefficient of variation of ASFV,CSFV,and APPV was less than 1%.Twenty-two virus samples were detected by the multiplex real-time PCR,compared with national standard diagnostic and patented method assay for CSF(GB/T 27540–2011),ASF(GB/T 18648–2020),and APPV(CN108611442A),respectively.The sensitivity of this triple real-time PCR for CSFV,ASFV,and APPV was almost the same,and the compliance results were the same(100%).A total of 451 clinical samples were detected,and the results showed that the positive rates of CSFV,ASFV,and APPV were 0.22% (1/451),1.3%(6/451),and 0%(0/451),respectively.This assay provides a valuale tool for rapid detection and accurate diagnosis of CSFV,ASFV,and APPV.

A New High-throughput Real-time PCR Assay for the Screening of Multiple Antimicrobial Resistance Genes in Broiler Fecal Samples from China

Objective Antimicrobial resistance(AMR)has become a global concern and is especially severe in China.To effectively and reliably provide AMR data,we developed a new high-throughput real-time PCR assay based on microfluidic dynamic technology,and screened multiple AMR genes in broiler fecal samples.Methods A high-throughput real-time PCR system with an new designed integrated fluidic circuit assay were performed AMR gene detection.A total of 273 broiler fecal samples collected from two geographically separated farms were screened AMR genes.Results The new assay with limits of detection ranging from 40.9 to 8,000 copies/reaction.The sensitivity rate,specificity rate,positive predictive value,negative predictive value and correct indices were 99.30%,98.08%,95.31%,99.79%,and 0.9755,respectively.Utilizing this assay,we demonstrate that AMR genes are widely spread,with positive detection rates ranging from 0 to 97.07%in 273 broiler fecal samples.bla CTX-M,bla TEM,mcr-1,fex A,cfr,optr A,and int I1 showed over 80%prevalence.The dissemination of AMR genes was distinct between the two farms.Conclusions We successfully established a new high-throughput real-time PCR assay applicable to AMR gene surveillance from fecal samples.The widespread existence of AMR genes detected in broiler farms highlights the current and severe problem of AMR.

Development of A Real-Time PCR Assay for Plasmodiophora brassicae and Its Detection in Soil Samples

A SYBR Green I real-time PCR assay was developed to detect and quantify Plasmodiophora brassicae ribosomal DNA（rDNA） and internal transcribed spacer（ITS）.A pair of primers PBF1/PBR1 was designed based on the conservative region of rDNA-ITS of P.brassicae.The positive plasmid pB12 was obtained and used as the template to create standard curve.The specificity,sensitivity,and reproducibility of real-time PCR were evaluated respectively.Naturally and artificially infested soil samples containing different concentrations of P.brassicae were detected.The results demonstrated that standard curve established by recombinant plasmid was shown a fine linear relationship between threshold cycle and template concentration.The melting curve was specific with the correlation coefficient of 0.995 and that the amplification efficiency was 93.8%.The detection limit of P.brassicae genomic DNA was approximately 40 copies per 25 μL.The sensitivity of the assay was at least 100-fold higher than conventional PCR.Only DNA from P.brassicae could be amplified and detected using this assay,suggesting the highly specific of this assay.The coefficient of variation was less than 3%,indicating the PCR method revealed high reproducibility.The detection limit in soil samples corresponded to 1 000 resting spores g-1soil.Bait plants were used to validate the real-time PCR assay.This developed real-time PCR assay allows for fast and sensitive detection of P.brassicae in soil and should be useful in disease management and pest interception so as to prevent further spread of P.brassicae.

Development of a Duplex Real-time PCR assay for Detection of Perkinsus and Marteilia refringens in Shellfish

[ Objective] To improve the accuracy and efficiency of the detection which used in Perkinsus sp and Marteilia refringens, and then short- en the detective cycle. [ Method] According to the gene sequence of Perkinsus sp and Marteilia refringens from gene bank, design two pairs of spe- cific primers and two TaqMan probes with different fluorophores labeled. Optimizing the reactive conditions and reagent concentration in order that establishing the duplex real-time PCR method for detecting Perkinsus sp and Marteilia refringens simultaneously. [ Result ] The sensitivity of the du- plex real-time PCR method which about Pertdnsus sp and Marteilia refringens is 40 template copies. After combine the templates of Perkinsus sp and Marteilia refringens with different concentrations, this method still could be detect this two protozoan efficiently and synchronously. [ Condudon] The es- tablished duplex real-time PCR method for detecting Perkinsus sp. and Marteilia refringens possesses lots of advantages, such as specific, sensitive, rapid, quantitative and reproducible, can be used for clinical detection of infection which was caused by Perkinsus sp. and Marteilia refringens.

Inter-laboratory Validation of Real-time PCR Assays for Goat-,Horse-and Donkey-derived Material in Meat Products

In this study,we performed an inter-laboratory collaborative ring trial to develop and validate specific TaqMan real-time PCR assays for goat-,horse-,and donkey-derived material in meat products.The performances of these assays in different environments and situations were comprehensively evaluated.This ring trial involved the participation of 12 laboratories in Europe and Asia.The results from the participating laboratories were analyzed to determine the specificity,accuracy,false positive rate,limit of detection(LOD),and probability of detection(POD)of the developed assays.Statistical analysis showed that the false positive and negative rates were zero,the LOD was five copies/reaction,and the laboratory standard deviation(σ_(L))was 0.30 for all three assays.Thus,the results demonstrate that the developed methods are robust and suitable for the detection and identification of goat-,horse-,and donkey-derived materials in meat products.

基于Real-time PCR法检测乳粉中牛源性成分定量研究

基于Real-timePCR建立了乳粉中牛源性成分相对定量检测方法,并对牛的特异性引物与探针进行了特异性、灵敏度和稳定性测试。通过模拟不同浓度牛乳粉与马乳粉混合样本,根据其△Ct值的函数关系进行线性拟合进而绘制标准曲线,建立乳粉中牛源性成分的相对定量检测。结果显示,该方法的最低检测限为0.00001 mg/mL,回收率为91.11%~119.2%,组间变异系数≤0.58%、组内变异系数≤1.44%。说明该方法在特异性与稳定性上适用于乳粉中牛源性成分及含量的掺假检测。

一种基于real-time PCR技术的TTV检测方法的建立及应用

目的:本研究旨在开发一种具有更高灵敏度和特异性的TTV检测技术,为揭示TTV在多种疾病过程中的作用提供重要的技术支持。方法:为了更精确、灵敏的检测TTV,本研究分析了目前公布的所有亚型的TTV基因序列,在此基础上建立了一种基于UTR区域的real-time PCR检测方法,并与文献报道应用较为广泛的PCR检测方法进行了对比。结果:本研究建立的方法在1×10^(7)~1×10^(1) copies/μL标准品浓度范围内具有良好的线性关系,相关系数为1.000,斜率为-3.446,检测下限为1×10^(1) copies/μL。重复性试验结果显示,组内变异系数为7.22%,表明本方法重复性、稳定性较强。针对30份临床样本,使用本研究建立的real-time PCR检测方法及目前被多个研究所使用的4套引物进行对比。结果表明,本研究所建立的方法灵敏度显著高于文献中报道的4种方法(P<0.01);Sanger测序结果表明,本方法检测出的30份阳性样本均为TTV,检测特异性为100%。结论:本研究采用基于TaqMan探针的real-time PCR检测方法,检测灵敏性高、覆盖基因型范围广,尤其对于TTV病毒载量较低的情况下能够进行定量检测,对于TTV病毒的致病性及作为免疫标志物的应用提供重要的技术支持。

Prevalence and Antibiotic Resistance of Urinary Tract Pathogens, with Molecular Identification of Klebsiella pneumoniae, Klebsiella oxytoca, and Acinetobacter spp., Using Multiplex Real-Time PCR

Urinary tract infections (UTIs) caused by uropathogens are a significant public health problem, and their treatment primarily relies on antibiotic therapy. However, the increasing global development of antibiotic resistance necessitates updating diagnostic techniques to ensure higher sensitivity and specificity, especially with advancements in science and medicine. This study aimed to evaluate the prevalence of UTIs and antibiotic resistance profiles through urine culture, as well as to identify Klebsiella pneumoniae, Klebsiella oxytoca, and Acinetobacter spp. in urine samples using a molecular approach with multiplex real-time PCR. From May 3 to July 25, 2023, at the Pietro Annigoni Biomolecular Research Center (CERBA) and Saint Camille Hospital of Ouagadougou (HOSCO), 209 urine samples collected from patients with suspected UTIs were analyzed using both urine culture and multiplex real-time PCR. Among the 209 patients, 52.15% were male and 47.85% female, with an average age of 46.87 ± 21.33 years. Urine cultures revealed an overall UTI prevalence of 23.44%, with a prevalence of 8.13% in men versus 15.31% in women (P = 0.023). The bacterial prevalence rates were as follows: Escherichia coli (12.92%), Klebsiella spp. (7.18%), Enterobacter cloacae (1.44%), Staphylococcus aureus (0.96%), and other bacteria. Klebsiella spp. demonstrated 100% resistance to Amoxicillin and Amoxicillin/Clavulanic Acid, while Escherichia coli showed 96.2% and 65.4% resistance to Amoxicillin and Amoxicillin/Clavulanic Acid, respectively. PCR analysis of the target bacteria revealed mono-infection prevalence rates of Klebsiella pneumoniae (10.39%), Klebsiella oxytoca (7.79%), and Acinetobacter spp. (7.79%), along with a co-infection prevalence rate of Klebsiella pneumoniae/Acinetobacter spp. (1.30%). This study demonstrated that PCR, with its high sensitivity and specificity, could effectively distinguish Klebsiella pneumoniae from Klebsiella oxytoca and detect Acinetobacter spp. in less than 24 hours—something urine culture alone could not achieve. The relative ease of automating urine PCR testing, combined with its diagnostic accuracy and rapid turnaround time, makes it a valuable addition to modern medical practice for the laboratory diagnosis of UTIs.

24重荧光real-time PCR技术在食物中毒快速检测中的应用

目的:应用24重荧光real-time PCR检测技术快速筛检食物中毒病原菌,结合国家标准中的培养法探讨24重荧光real-time PCR检测技术符合性和应用价值。方法:采用高灵敏度、高特异性的24重荧光real-time PCR检测技术作为中毒病原菌的初筛方法,国标方法进行细菌分离培养,并对分离出的病原菌进行生化鉴定。结果:5份食物中毒患者肛拭子在增菌前检出4份霍乱弧菌核酸(非O1/非O139群,24重荧光real-time PCR),9份患者肛拭子在增菌后检出5株霍乱弧菌(非O1/非O139群,国标培养法),其中包含4份PCR技术初筛阳性样品,两个方法的符合率为80%。所有样本均未检出沙门氏菌、志贺菌、副溶血性弧菌、致泻性大肠埃希菌以及金黄色葡萄球菌。结论:应用24重荧光real-time PCR检测技术同时检测24种常见致病病原菌,能高效锁定中毒病原菌。将其与国标培养法相结合,对临床治疗和食物中毒快速处置能起到积极作用,值得应用和推广。

A Duplex Real-Time PCR Assay for the Simultaneous Detection of Porcine Circovirus 2 and Circovirus 3

Porcine circoviruses(PCV) include PCV1, PCV2, and the new-emerging PCV3. PCV2 is pathogenic to pigs, but the pathogenicity of PCV3 in pigs is debatable. Recently, there have been frequent reports of PCV2 and PCV3 co-infections in clinical samples. Thus, it would be practical to develop a duplex PCR method to detect PCV2 and PCV3 simultaneously. In this study, specific primers and probes were designed to target PCV2 cap and PCV3 rep genes. A duplex real-time PCR method was then developed to detect the two viruses. The assay was found to be highly specific, sensitive, and reproducible for PCV2/3 without cross-reactions with other swine pathogens. The sensitivity of this assay was 2.9 copies for the PCV2 plasmid and 22.5 copies for the PCV3 plasmid. The established assay was then used to detect PCV2/3 infection in 340 clinical samples collected in the first half of 2017. The results showed that the co-infection rate of PCV2/3 in the samples was 27.6%. Our study provides an important tool that can be used to perform urgently needed surveys for the two porcine circoviruses to evaluate their impact on the swine industry.

Real-time RT-PCR Assay for the Detection of Culex flavivirus

Based on the Culex flavivirus (CxFV) E gene sequences in GenBank, CxFV-specific primers and probes were designed for real-time reverse transcription-polymerase chain reaction (RT-qPCR). The specificity test revealed that CxFV could be detected using RT-qPCR with the specific CxFV primers and probes; other species of arboviruses were not detected. The stability test demonstrated a coefficient of variation of <1.5%. A quantitative standard curve for CxFV RT-qPCR was established. Quantitative standard curve analysis revealed that the lower detection limit of the RT-qPCR system is 100 copies/mu L. Moreover, RT-qPCR was used to detect CxFV viral RNA in mosquito pool samples. In conclusion, we established a real-time RT-PCR assay for CxFV detection, and this assay is more sensitive and efficient than general RT-PCR. This technology may be used to monitor changes in the environmental virus levels.

Real-time RT-PCR Assay for the detection of Tahyna Virus

A real-time RT-PCR （RT-qPCR） assay for the detection of Tahyna virus was developed to monitor Tahyna virus infection in field-collected vector mosquito samples. The targets selected for the assay were S segment sequences encoding the nucleocapsid protein from the Tahyna virus. Primers and probes were selected in conserved regions by aligning genetic sequences from various Tahyna virus strains available from GenBank. The sensitivity of the RT-qPCR approach was compared to that of a standard plaque assay in BHK cells. RT-qPCR assay can detect 4.8 PFU of titrated Tahyna virus. Assay specificities were determined by testing a battery of arboviruses, including representative strains of Tahyna virus and other arthropod-borne viruses from China. Seven strains of Tahyna virus were confirmed as positive; the other seven species of arboviruses could not be detected by RT-qPCR. Additionally, the assay was used to detect Tahyna viral RNA in pooled mosquito samples. The RT-qPCR assay detected Tahyna virus in a sensitive, specific, and rapid manner; these findings support the use of the assay in viral surveillance.

Development of two multiplex real-time PCR assays for simultaneous detection and differentiation of monkeypox virus IIa,IIb,and I clades and the B.1 lineage

An ongoing multi-country outbreak of monkeypox was reported in May 2022 with several deaths,affecting 107 countries of all six World Health Organization(WHO)regions.The WHO has declared the current monkeypox outbreak to be a Public Health Emergency of International Concern.It is,thus,necessary to rapidly and accurately detect and distinguish different monkeypox virus(MPXV)clades.We designed primers and probes based on the alignment of 138 complete genomes of poxviruses.In Panel 1,we mixed one pair of primers and three probes to detect and differentiate the MPXV Western Africa(IIa,IIb clade)and Congo Basin(I clade)and other orthopoxviruses.In Panel 2,we mixed one pair of primers and two probes to detect the 2022 MPXV(B.1 lineage and its descendant lineages).In addition,we tested the specificity and sensitivity of the assay using real-time PCR.In Panel 1,the assay reproducibly identified various concentrations of two plasmids of the monkeypox virus,whereas other orthopoxviruses did not cross-react.In Panel 2,the probe annealed well to MPXV B.1 and showed the expected linearity.These two multiple real-time assays are inclusive and highly specific for identifying different clades of MPXV.

Establishment of TaqMan Real-time Quantitative PCR Assay for Foreign Gene Copy Numbers in Transgenic Soybean

TaqMan quantitative PCR technique was used to detect the copies of exogenous CaMV35S flanks sequence in transgenic soybean. With soybean lectin as the endogenous reference gene, and gene complex DNA in non-GMO soybeans as the endogenous reference standard, the gradient dilution method was used to separately calculate Ct value of endogenous reference gene and plasmid DNA and correlation standard curve equation of logarithm of copies, and then to calculate the copies of samples through substituting thus-obtained Ct into the standard curve equation. The standard curve equation of endogenous reference gene was y =–3.422x＋35.201, R2=0.998; the standard curve equation of exogenous gene was y =–3.495x＋35.303, R2=0.999. The sample copies was got by putting Ct value into the standard curve equation, and it was the ratio of exogenous gene and reference gene. We found that CaMV35S gene in transgenic soy was single copy.

MGB probe assay for rapid detection of mtDNA11778 mutation in the Chinese LHON patients by real-time PCR

Objective： Leber＇s hereditary optic neuropathy （LHON） is a maternally inherited degeneration of the optic nerve caused by point mutations of mitochondrial DNA （mtDNA）. Many unsolved questions regarding the penetrance and pathophysiological mechanism of LHON demand efficient and reliable mutation testing. This study aims to develop a minor groove binder （MGB） probe assay for rapid detection of mtDNA11778 mutation and heteroplasmy in Chinese LHON patients by real-time polymerase chain reaction （PCR）. Methods： Forty-eight patients suspected of having LHON and their maternal relatives underwent a molecular genetic evaluation, with 20 normal individuals as a control group at the same time. A real-time PCR involving two MGB probes was used to detect the mtDNA 1 1778 mutation and heteroplasmy. A linear standard curve was obtained by pUCmLHONG and pUCmLHONA clones. Results： All 48 LHON patients and their maternal relatives were positive for rntDNA11778 mutation in our assay, 27 heteroplasmic and 21 homoplasmic. Eighteen cases did not show an occurrence of the disease, while 9 developed the disease among the 27 heteroplasmic mutation cases. Eleven did not show an occurrence of the disease, while 10 cases developed the disease among 21 homoplasmic mutation cases. There was a significant difference in the incidence between the heteroplasmic and the homoplasmic mutation types. The time needed for running a real-time PCR assay was only 80 min. Conclusion： This real-time PCR assay is a rapid, reliable method for mtDNA mutation detection as well as heteroplasmy quantification. Detecting this ratio is very important for predicting phenotypic expression of unaffected carriers.

Establishment of a Real-time Fluorescent Quantitative PCR Assay for Detection of Genetically Modified Maize Line MON88017

In order to improve the standardized technical systems of quantitative analyses for genetically modified organisms （GMOs） and products, ensure bio-safety and reduce ecological risk in China, a real-time fluorescent quantitative PCR assay was established for detection of genetically modified maize line MON88017. The established method was evaluated based on the specificity, sensitivity, accuracy and measurement uncertainty. The results showed that the established method had strong specificity in detection of genetically modified maize line MON88017. 1.50% MON88017 sample was detected with 29 replica- tions. The average measured value （ 1. 541% ） was close to the actual value （ 1.50% ） and the relative deviation was 2.70%. The variation coefficient of the measured value was 0.110 g ; the recovery was 100.00% and the measurement uncertainty was 0. 096. The limit of detection for genetically modified maize line MON88017 with the established method was 5 copies at the 97.5% confidence level. Thus, the real-time fluorescent quantitative PCR assay established in this study exhibited high specificity, accuracy and sensitivity, which could provide technical support for the safety supervision of genetically modified organ- isms and products in China.