The current accurate quantitative technology, such as standard curve-based quantitative real time PCR (qPCR) and digital PCR (dPCR), is time-consuming and expensive, resulting in high costs for the implementation of the GMO labeling policy. This study proposed a rapid quantitative strategy that combines the 2-Delta Delta Ctmethod with the t-test of Delta Ct values between the test samples and the reference control. This method allowed for an approximate estimation of the GMO content of test samples while identifying the GM events. GMO labeling or exempt for most samples can be determined based on the rapid estimation of GMO content and the t-test result of the Delta Ct values between the test samples and the reference control. This approach streamlines the quantitative analysis process, makes the quantification more accessible and affordable, and supports compliance with GMO labeling regulations worldwide.

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas proteins coupled with preamplification have shown great potential in molecular diagnoses. However, the current CRISPR-based methods require additional reporters and time-consuming process. Herein, a gold nanoparticle (AuNP)enhanced CRISPR/dCas9-mediated fluorescence resonance energy transfer (FRET) termed Au-CFRET platform was proposed for rapid, sensitive, and specific detection of nucleic acid for the first time. In the Au-CFRET sensing platform, AuNP was functionalized with dCas9 and used as nanoprobe. Target DNA was amplified with FAM-labeled primers and then precisely bound with AuNP-dCas9. The formed complex rendered the distance between AuNP acceptor and FAM donor to be short enough for the occurrence of FRET, thus resulting in fluorescence quenching. Moreover, AuNPs were demonstrated to enhance binding efficiency of dCas9 to target DNA in Au-CFRET system. The key factors regarding the FRET efficiency were analyzed and characterized in detail, including the length of donor/acceptor and the size of AuNPs. Under the optimal conditions, Au-CFRET could determinate CaMV35S promoter of genetically modified rice as low as 21 copies mu L-1. Moreover, AuCFRET sensing system coupled with one-step extraction and recombinase polymerase amplification can identify the genuine plant seeds within 30 min from sampling to results at room/body temperature without expensive equipment or technical expertise, and requires no additional exogenous reporters. Therefore, the proposed sensing platform significantly simplified the system and shortened the assay time for nucleic acid diagnoses.

Wearable sensors with multiple functions are attracting significant attention due to their broad applications in health monitoring and human-computer interaction. Despite significant progress in wearable sensors, it is a significant challenge to monitor temperature and stress simultaneously with a single sensor. A wearable multifunctional optical sensor based on Er3+/Yb3+ co-doped Gd2O3 nanoparticles and a tapered U-shaped fiber is proposed to monitor both temperature and stress in this paper. Temperature resolution of about 0.16 degrees C is achieved by monitoring the fluorescence intensity ratio (FIR) around 562 nm and 522 nm emitted by Er3+/Yb3+ co-doped Gd2O3 phosphors, which are integrated in a single-mode fiber (SMF). The stress measurement is obtained by monitoring the fluorescence intensity change around 522 nm, which is insensitive to temperature. The results show that the pressure sensitivity and low detection limit are 7% kPa-1 and 127 Pa, respectively. In addition, the response time of 20 ms are achieved for stress sensing. As a proof-of-concept, human skin temperature and heart and respiratory rates are detected before and after exercise by positioning the sensing probe on the wrist. Furthermore, heart and respiratory rates in different parts of the body are also monitored, which are in good agreement with one another. The results demonstrate that the proposed wearable multifunctional optical sensor has huge potential for health monitoring. (c) 2025 Optica Publishing Group. All rights, including for text and data mining (TDM), Artificial Intelligence (AI) training, and similar technologies, are reserved.

The genetically modified (GM) maize DBN9936 with a biosafety certificate will soon undergo commercial application. To monitor the safety of DBN9936 maize, three genomic DNA (gDNA) reference materials (RMs) (DBN9936a, DBN9936b, and DBN9936c) were prepared with nominal copy number ratios of 100%, 3%, and 1% for the DBN9936 event, respectively. DBN9936a was prepared from the leaf tissue gDNA of DBN9936 homozygotes, while DBN9936b and DBN9936c were prepared by the quantitative mixing of gDNA from the leaf tissues of DBN9936 homozygotes and non-GM counterparts. Validated DBN9936/zSSIIb duplex droplet digital PCR was demonstrated to be an accurate reference method for conducting homogeneity study, stability study, and collaborative characterization. The minimum intake for one measurement was determined to be 2 μL, and the gDNA RMs were stable during transport at 37 °C for 14 days and storage at −20 °C for 18 months. Each gDNA RM was certified for three property values: DBN9936 event copy number concentration, zSSIIb reference gene copy number concentration, and DBN9936/zSSIIb copy number ratio. The measurement uncertainty of the certified values took the uncertainty components related to possible inhomogeneity, instability, and characterization into account. This batch of gDNA RMs can be used for calibration and quality control when quantifying DBN9936 events.

The genetically modified (GM) soybean DBN8002 has been approved for commercial planting in China. For enforcing GMO labeling policy, an event-specific real-time quantitative PCR (qPCR) method was developed to target the junction fragment between the T-DNA left border and the flanking genomic DNA, yielding a 104 base pair (bp) amplicon. This event-specific qPCR method can identify and quantify the DBN8002 event with high specificity, satisfactory linearity, and acceptable accuracy. Furthermore, the DBN8002-event specific primer/probe set was successfully transferred to a droplet digital PCR (ddPCR) platform for quantification. The quantitative results from qPCR were found to be comparable to those obtained from ddPCR, with a P-value exceeding 0.05, indicating no significant difference. The limit of detection (LOD) for both qPCR and ddPCR methods was determined to be 10 copies per reaction, while the limit of quantification (LOQ) was estimated to be 20 copies per reaction for qPCR and 40 copies per reaction for ddPCR. The collaborative validation demonstrated that the DBN8002 event-specific qPCR method had satisfactory repeatability and reproducibility. Both the event-specific qPCR and ddPCR methods are suitable for quantifying the DBN8002 content in samples. Additionally, ddPCR can be utilized for the characterization of DBN8002 reference materials.

[Objective]The enforcement of labeling regulations on genetically modified organisms(GMOs)requires establishing a standard system for accurate quantification of GMOs that includes the standard or guide for estimating measurement uncertainty(MU).It is urgent to establish a standardized top-down approach for estimating MU of quantitative results,which is conveniently adopted by the general testing laboratories.[Method]There are two approaches for estimating the MU introduced by precision of quantitative method,one is to establish the equation of MU estimation using data obtained on 15 routine samples based on the"uncertainty function",the other is to evaluate the MU by repeatedly measuring a certified reference material(CRM)and calculating the intermediate precision.The uncertainty introduced by bias is evaluated using a CRM or a sample prepared by laboratory as bias control.The uncertainty of the nominal value of the sample prepared by laboratory is evaluated by using a simplified program based on the preparation process.The MU contributed by method precision and bias are combined into the standard uncertainty of the quantitative results,and then multiplied by the coverage factor k to obtain the expanded uncertainty.[Result]The event-specific PCR method of genetically modified maize DBN9936 was took as an example.The MU of method precision was evaluated to be 0.76%using simulated DBN9936 routine samples,and to be 0.33%using a CRM(GBW(E)100901).Compared with routine samples,the MU of method precision evaluated using a CRM is significantly underestimated.The uncertainty introduced by bias was evaluated to be 0.26%using a CRM(GBW(E)100901)as a bias control.Using a laboratory prepared powder sample and a genomic DNA sample(nominal values of 3.0%)as bias control,the bias uncertainty was evaluated to be 0.20%and 0.19%,respectively.Since the simplified program ignored some uncertainty components,the uncertainty of the nominal value of laboratory prepared samples was estimated to be smaller.By combining the MU of method precision and bias,the expanded uncertainty using routine samples was obtained to be 1.26%,1.20%,and 1.20%,respectively,the expanded uncertainty using a CRM was 0.84%,0.78%,and 0.76%,respectively.[Conclusion]This study established the top-down approaches for MU estimation of quantitative results,testing laboratories should prioritize routine samples to estimate the MU contributed by the method precision,and select CRMs as bias control in principle to evaluate bias uncertainty during GMO quantification.

Transgenic maize Ruifeng 125 is one of the first series of transgenic maize varieties requesting safety certificate in China. To improve the supervise efficiency by rapid detection for Ruifeng 125, recombinase polymerase amplification（RPA） technology was developed. RPA primes were designed according to the junction border between insertion DNA and the host genome. Meanwhile, the screening procedure, reaction temperature, time and primer concentration were also optimized. Results showed that developed RPA method had a wide range of temperature from 30℃ to 45℃, and could be implemented within 20 min, showing less time cost than PCR. The best primer concentration of 0.35 μmol/L was adopted for obtaining satisfactory assay performance and better amplification efficiency. The results demonstrated that the PRA method was capable for rapid amplification of Ruifeng 125 with high specificity and sensitivity. The detection limit of this method was 36 copies. This work provides a promising platform for the detection of Ruifeng 125 or other commercial transgenic crops, and expects to develop of point-of-care identification.

【Objective】The GMO（genetically modified organism） reference materials（RMs） are the material basis for GMO safety supervision and labeling policy implementation. During GMO detection the utilization of RMs guarantees the traceability of quantitative results within laboratory and the comparability of quantitative results between laboratories. GM（genetically modified）maize MON87427 has been approved to be imported as raw material in China, it’s urgent to develop certified reference materials（CRMs） for safety supervision and quantification. 【Method】 The GM maize MON87427 hybrid seeds and non-GM counterparts provided by the developer were used as raw materials to perform washing, drying, freeze-grinding, particle size measurement, and moisture content measurement, sequentially. The matrix RMs of MON87427a, MON87427b and MON87427c were produced by blending the seed powder of the GM maize MON87427 and a non-GM counterpart in GMO mass fractions of 60.0 mg·g-1, 99.5mg·g -1 and 1 000.0 mg·g -1 on a dry basis. The real-time quantitative PCR was used to conduct an initial assessment of homogeneity before packing. The MON87427/z SSIIb duplex digital PCR（ddPCR） was used to evaluate the homogeneity and stability of the RMs as well as the collaborative characterization by 8 qualified laboratories. The data processing of homogeneity, stability, collaborative characterization together with uncertainty evaluation of RMs were carried out according to the standard "General and Statistical Principles for Characterization of Reference Materials"（JJF 1343）. 【Result】 This batch of RMs contains three RMs of MON87427a,MON87427b, and MON87427c in different mass fractions, with more than 80% of particle size of less than 200 μm and less than 5%of moisture content. The RMs were packed in brown glass bottles, nitrogen flushed before capping, bottling amount was not less than 1.0 g/bottle, a total of 400 bottles were produced for each mass fraction RM. The calculated F values of the homogeneity test were all less than the critical value of F 0.05（14, 30） （2.04） for the three RMs, displaying good homogeneity within and between bottles, and the minimum intake was determined to be 100 mg. The RMs can be stored stably at 25℃, 37℃, and 60℃ for 14 days, the property value of the RMs does not change significantly after 14 days of transportation at room temperature; the long-term stability can reach 12 months at 4℃ and-20℃; The property value of the samples taken from the same bottle of RM after 5 opening-capping cycles,does not deviate significantly from that of the first taken sample. The collaborative characterization data by eight qualified laboratories displayed normal distribution without outliers and outlying standard deviations. The standard value and expanded uncertainty of MON87427a, MON87427b, Mon87427c were certified to be（2.92±0.44）%,（4.89±0.57）%,（52.1±3.4）%.【Conclusion】The developed GM maize MON87427 matrix RMs in different mass fractions have good homogeneity, and can be stably transported and stored. This batch of RMs meets the requirements of qualitative and quantitative detection of MON87427event, providing reliable CRMs for the safety supervision and implementation of quantitative labeling policy for GMO-derived products.

Specific and economical nucleic acid detection is crucial for molecular diagnoses in resource-limited settings. Various facile readout approaches have been developed for nucleic acid detection, but they have limited specificity. Herein, nuclease-dead Cas9 (dCas9)/sgRNA was used as an excellent DNA recognition probe system to develop a visual clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9-mediated enzyme-linked immunosorbent assay (ELISA) for specific and sensitive detection of cauliflwer mosaic virus 35s (CaMV35S) promoter in genetically modified (GM) crops. In this work, the CaMV35S promoter was amplified with biotinylated primers, and then precisely bound with dCas9 in the presence of sgRNA. The formed complex was captured by antibody-coated microplate and bound to a streptavidin-labeled horseradish peroxidase probe for the visual detection. Under the optimal conditions, dCas9-ELISA could detect CaMV35s promoter as low as 12.5 copies μL-1. Moreover, the proposed method was capable to distinguish the target sequence with single-base specificity. Coupled with one-step extraction and recombinase polymerase amplification, dCas9-ELISA can identify actual GM rice seeds within 1.5 h from sampling to results without expensive equipment and technical expertise. Therefore, the proposed method offers a specific, sensitive, rapid and cost-effective detection platform for molecular diagnoses.

In polymerase chain reaction (PCR)-based nucleic acid quantification, the DNA template type, primer/probe sequence, and instrument platform such as real-time quantitative PCR (qPCR) and digital PCR (dPCR) affect the accuracy and reliability of quantitative results. In this study, a plasmid DNA (pDNA) pBI121-screening, genetically modified (GM) rice SDrice genomic DNA (gDNA), and GM rapeseed SDrape gDNA, all carrying the same 11 screening elements, were used to prepare samples of different levels of gDNA and pDNA in a non-GM gDNA background. The comparison of the dPCR assays targeting the 11 screening elements revealed that the primer/probe set is a key factor that affects the accuracy of dPCR quantification. The optimal PCR method for the 11 screening elements was screened out from among the validated qPCR methods. The accuracy of the qPCR quantification of the low-level pDNA and gDNA test samples was low when pDNA was used as a calibrator, whereas that of the dPCR quantification was high and not affected by variations in template type and detection target. The validated dPCR assays targeting one or two elements can be randomly selected to characterize multiple-target pDNA reference materials (RMs). Low-level pDNA RMs with certified values can be used as quality controls for dPCR assays to avoid significant bias in gDNA quantification.

The hygromycin phosphotransferase (HPT) gene as a selective marker is normally used in screening tests as a first step in detecting and quantifying genetically modified organisms (GMOs) in seeds, food, and feed materials. Nevertheless, if researchers only focus on the HPT gene, it is difficult to distinguish genetically modified (GM) crops from microbial infection, leading to miscalculation of the rate of GM materials in a given sample set. Here, we cloned the 7259 bp sequence carrying the HPT gene from soybean sprouts using the genome walking strategy. BLAST analysis revealed that this sequence was derived from plasmids naturally occurring in microorganisms, such as Escherichia coli, Klebsiella pneumoniae or Salmonella sp. Using the reconstructed plasmid pFP-hpt, qualitative PCR and quantitative real-time PCR (qPCR) methods were established, and 261 bp and 156 bp products were produced. The specificity of these assays was assessed against related pFP-hpt plasmids, plant species with important agronomic traits, and GM crops containing the HPT gene. No unexpected results were observed between samples using these qualitative PCR and qPCR methods. The sensitivity of this qualitative PCR assay was determined at 20 copies, while the limit of detection (LOD) and limit of quantification (LOQ) of qPCR were both 5 copies per reaction. Our in-house validation indicated that the amplification efficiency, linearity, and repeatability of this qPCR assay were in line with performance requirements. Furthermore, a qualitative and quantitative duplex PCR showed high reliability for the simultaneous detection of the HPT gene in a plant sample and environmental micro-organisms harboring the HPT gene in one PCR reaction. These qualitative PCR and qPCR assays were able to differentiate between plants infected with E. coli harboring the HPT gene from GM plants, indicating that these two methods are broadly applicable for routine GMO testing.

CP4-EPSPS (Agrobacterium sp. strain CP4 5-enolpyruvylshikimate-3-phosphate synthase) protein showed remarkable thermostability and was highly resistant to proteases, such as trypsin. In order to eliminate the pollution of CP4-EPSPS from the accumulated straws to the surrounding environment during the winter, the present study investigated the extracellular proteases of 21 psychrophilic strains isolated from the south polar region. The results indicated that Stenotrophomonas maltophilia 780 was able to degrade CP4-EPSPS at 18 °C efficiently. Further study indicated that it was able to grow in the extract of Roundup Ready soybean at 18 °C, with CP4-EPSPS degraded to an undetectable level within 72 h. The extracellular proteases of Stenotrophomonas maltophilia 780 are thermo-sensitive, with an optimal temperature of 65 °C. The genomic sequencing result indicated that this strain had more than a hundred putative protease and peptidase coding genes, which may explain its high capability in decomposing CP4-EPSPS.

The genetically modified (GM) maize DBN9936 that has been granted a biosafety certificate needs to be regulated in China. An event-specific PCR assay targeting the junction region between the left border of its T-DNA and the flanking genomic DNA (gDNA) was established to be able to specifically identify the DBN9936 event from other GM maize and non-GM maize. Both qPCR and duplex ddPCR achieved accurate measurement of DBN9936 content in blind matrix and gDNA samples with acceptable precision in terms of a relative standard deviation (RSD) of <25 % and a trueness in terms of relative bias of +/- 25 %. The quantitative values of the same sample did not show significant difference between qPCR and duplex ddPCR, whereas the duplex ddPCR showed advantage over qPCR in terms of precision. This study provided an event-specific PCR method for identification and quantification of DBN9936 on both a real-time PCR platform and a ddPCR platform.

[目的]批准进口的转基因玉米NK603是中国转基因生物安全监管的主要对象.转基因安全监管需要标准物质和标准检测方法,建立转基因玉米NK603的数字PCR (dPCR)方法将为转基因玉米NK603的定量检测和标准物质研制提供精准测量技术.[方法]采用人工合成技术构建标准质粒分子pUC57-NK603;将NK603转化体与不同的玉米内标基因引物/探针一一组合,遴选与NK603转化体特异性PCR具有相同扩增能力的玉米内标基因PCR方法;设置二重微滴数字PCR(ddPCR)的退火温度梯度和引物/探针浓度梯度,优化二重ddPCR的反应体系和反应条件;用梯度稀释的标准质粒溶液作模板,考察二重ddPCR的检测极限、定量极限和动力学范围;将转基因玉米NK603种子粉末和非转基因玉米种子粉末混合,配制质量分数分别为100％、10％和6％的盲样,考察二重ddPCR的定量准确性.[结果]用标准质粒分子pUC57-NK603作为二重ddPCR的质控对照,通过考察二重ddPCR反应热图的微滴信号强度、阳性微滴与阴性微滴分辨率、雨滴数量、NK603转化体与内标基因拷贝数比值测量值与预期值的一致性,确定将NK603转化体特异性PCR方法与内标基因zSSⅡb PCR方法组合,建立NK603/zSSⅡb二重ddPCR方法.二重ddPCR反应体系中NK603转化体和zSSⅡb内标基因的引物/探针浓度相同,均为400 nmol.L-1/200 nmol·L-1,在60℃退火延伸.NK603/zSSⅡb二重ddPCR的检测极限是2 copies DNA模板,定量极限是48 copies DNA模板,动力学范围是10-60000 copies DNA.应用NK603/zSSⅡb二重ddPCR方法可准确定量玉米盲样中的NK603转化体含量,定值结果变异系数小于25％;dPCR定量结果与荧光定量PCR (qPCR)定量结果无显著差异,且具有更高的精确性.[结论]内标基因的选择会影响dPCR定量结果的准确性,在建立dPCR方法的过程中,要用具有准确量值的样品作为质控对照,评估内标准基因的适用性.以人工合成的标准质粒分子pUC57-NK603为质控对照,建立了NK603/zSSⅡb二重ddPCR方法.应用建立的二重ddPCR定值方法进行标准物质的研制和定值,已成功研制出转基因玉米NK603有证标准物质.

随着转基因技术发展和应用,转基因外源基因表达蛋白在环境中富集和降解情况成为公众关注的问题,外源蛋白在环境中的检测和监测成为转基因研究和监管的一个方向.本研究利用模拟自然降解和蛋白酶K两种方式,处理转基因大豆种子(5％GTS 40-3-2),采用Western blot、双抗夹心快速检测试纸条和酶联免疫吸附测定(ELISA)等手段检测CP4-EPSPS蛋白降解情况,结果发现在模拟自然降解条件下,CP4-EPSPS蛋白呈现逐渐被降解的趋势,且在17周被彻底降解;蛋白酶K处理大豆匀浆情况下,9小时CP4-EPSPS蛋白被快速降解.该研究探索了CP4-EPSPS蛋白在自然环境下的降解规律,为寻求一种快速、安全、有效降解转基因CP4-EPSPS蛋白研究提供参考.

实施转基因产品定量标识制度需要建立准确可靠的定量检测技术和方法.数字PCR(dPCR)不依赖标准物质,实现对DNA分子的绝对定量,已成功用于转基因含量检测和标准物质定值.为建立可靠的dPCR方法,获得准确测量结果,本研究以耐除草剂玉米MON87427为材料,探索建立二重微滴数字PCR(ddPCR)的策略.以构建的聚合MON87427转化体和5个玉米内标基因的重组质粒pUC57-M为质控样品,将5个不同的玉米内标基因分别与MON87427组合,通过优化退火温度,根据阳性微滴与阴性微滴分辨率、中等信号强度雨滴数量及测量值与理论值的一致性等,确定了二重ddPCR组合为MON87427/zSSIIb,最适退火温度为58.4℃.MON87427/zSSIIb二重ddPCR的动力学范围为10～60000拷贝.对盲样进行定量检测,二重ddPCR的定量结果与荧光定量PCR有良好的可比性,表明MON87427/zSSIIb二重ddPCR可取代qPCR方法进行转基因玉米MON87427的定量检测及标准物质定值.

现场快速检验(point of care test,POCT)是实现转基因基层监管必要的技术手段,在转基因成分检测中发挥着重要作用.生物传感器为转基因成分简单、快速和低成本定量分析提供了一个可行的技术方案.目前大量的生物传感器研究还处于方法学研究阶段,研究者需要着重将生物传感器技术与分析设备集成,开发小型化的便携式检测系统,从而满足POCT分析.本文回顾了近年来已开发的基于手持式设备的用于转基因POCT分析的微型生物传感器系统研究进展,提出了未来可用于转基因POCT分析的生物传感器的小型化策略,展望了小型化转基因生物传感器系统的发展趋势、发展前景和面临的挑战.

为了对基因组编辑产品进行精准定性和定量检测,以水稻SP1基因的编辑植株为材料,在编辑位点上下游设计通用引物,在编辑位点处设计基因编辑位点特异性TaqMan探针,建立了编辑位点特异性PCR方法.利用该方法可准确鉴定特异基因组编辑产品,检测灵敏度达到5～10拷贝,可在实时荧光PCR(qPCR)和微滴数字PCR(ddPCR)平台上对基因组编辑产品进行定量检测.由于数字PCR的微反应单元可消除野生型DNA对通用引物的竞争性消耗,与qPCR的定量结果相比,ddPCR定量结果具有更高的定量准确性.

转基因油菜是我国转基因生物安全监管的重要对象,为了解决检测机构常常面临的标准物质(样品)缺乏困境,统计我国批准进口和正在申请安全证书的11个转基因油菜品种,分析分子特征和相应的检测标准方法,确定每个转基因油菜品种的检测靶标序列.将11个转基因油菜品种的检测靶标序列通过基因合成的方法融合构建到pUC18载体上,研制出阳性质粒分子pYCID-1905,为转基因油菜的转化体鉴定检测提供了通用的阳性对照分子.结果发现,该质粒分子可同时用作国家标准(GB/T)、农业农村部公告、进出口检验检疫标准(SN/Y)和欧盟标准中普通PCR方法和实时荧光PCR方法的阳性对照,可以解决转基因油菜检测中缺乏标准样品的难题.

Genome-edited plants created by genome editing technology have been approved for commercialization. Due to molecular characteristics that differ from classic genetically modified organisms (GMOs), establishing regulation-compliant analytical methods for identification and quantification of genome-edited plants has always been regarded as a challenging task. An editing-site-specific PCR method was developed based on the unique edited sequence in CAO1-edited rice plants. Test results of seven primer/probe sets indicated that this method can identify specific CAO1-edited rice from other CAO1-edited rice and wild types of rice with high specificity and sensitivity. The use of LNA (locked nucleic acid) in a probe can efficiently increase the specificity of the editing-site-specific PCR method at increased annealing temperature which can eliminate non-specific amplification of the non-target. The genome-edited ingredient content in blinded samples at the level of 0.1% to 5.0% was accurately quantified by this method on the ddPCR platform with RSD of <15% and bias in the range of ±17%, meeting the performance requirements for GMO detection method. The developed editing-site-specific PCR method presents a promising detection and quantification technique for genome-edited plants with known edited sequence.