A duplex qPCR assay for human erythropoietin (EPO) transgene to control gene doping in horses

The misuse of genetic manipulation technology to enhance athletic performance is termed gene doping which is prohibited in human sports, horseracing, and equestrian sports. Although many qPCR assays have been developed, most assays employ genomic DNA (gDNA) from humans, non-human primates, and mice as a background and they may not be applicable for testing horse samples. This study aimed to develop a qPCR assay for the detection of human erythropoietin (hEPO) transgene in horse blood cells where the viral vectors used in gene therapy can reside for months. For the detection of hEPO transgene, the performance of three sets of primers and a hydrolysis probe for hEPO were compared. One set showed adequate specificity, sensitivity, amplification efficiency, and a dynamic range of detection in the presence of horse gDNA. The assay was duplexed with the detection of horse tubulin α 4A (TUBA4A) gene as an endogenous internal control in order to prevent false-negative results due to poor recovery and storage of extracted DNA and/or qPCR experimental variation. For the extraction of hEPO-plasmid, the QIAGEN Gentra Puregene blood kit was shown to recover the majority (62%) of hEPO-plasmid from spiked horse blood cells. The specificity and limit of detection (LOD) of the duplex qPCR assay were determined in accordance with MIQE guidelines. These findings supported the application of this duplex qPCR assay to the detection of hEPO transgene in horse blood cells.     

Leishmania mini-exon genes for molecular epidemiology of leishmaniasis in China and Ecuador

The mini-exon gene is unique and is tandemly repeated in the Leishmania genome. The transcribed region is highly conserved, but the non-transcribed spacer region is distinct in length and in sequence among different Leishmania species. The usefulness of PCR amplification of the Leishmania mini-exon gene was examined for molecular epidemiology of visceral and cutaneous leishmaniasis. We previously described a PCR method for amplification of the mini-exon gene and obtained positive amplification in bone marrow aspirates of patients with visceral leishmaniasis in China. In this study, we have cloned and sequenced two PCR products from the patients. The sequences of two products revealed 100% identity and showed more similarity to the mini-exon gene of L. donovani Indian strain than those of L. donovani complex in Africa and South America. We also applied this PCR method to the diagnosis of cutaneous leishmaniasis. We obtained positive PCR amplification in skin biopsy materials taken from patients with cutaneous leishmaniasis in Ecuador. Since this PCR amplification is simple and requires only a pair of primers to detect all Leishmania species distributed in Ecuador, the method may be a useful tool for the detection of parasites, not only from patients, but also from sandflies and reservoir animals in this area of endemicity.     

Robustness of digital PCR and real-time PCR against inhibitors in transgene detection for gene doping control in equestrian sports

Gene doping is a threat to fair competition in sports, both human and equestrian. One method of gene doping is to administer exogenous genetic materials, called transgenes, into the bodies of postnatal humans and horses. Polymerase chain reaction (PCR)-based transgene detection methods such as digital PCR and real-time PCR have been developed for gene doping testing in humans and horses. However, the significance of PCR inhibitors in gene doping testing has not been well evaluated. In this study, we evaluated the effects of PCR inhibitors on transgene detection using digital PCR and real-time PCR against gene doping. Digital PCR amplification was significantly inhibited by high concentrations of proteinase K (more than 0.1 μg/μl), ethylenediaminetetraacetic acid (more than 5 nmol/μl), and heparin (more than 0.05 unit/μl) but not by ethanol or genomic DNA. In addition, phenol affected droplet formation in the digital PCR amplification process. Real-time PCR amplification was inhibited by high concentrations of phenol (more than 1% v/v), proteinase K (more than 0.001 μg/μl), ethylenediaminetetraacetic acid (more than 1 nmol/μl), heparin (more than 0.005 unit/μl), and genomic DNA (more than 51.9 ng/μl) but not by ethanol. Although both PCR systems were inhibited by nearly the same substances, digital PCR was more robust than real-time PCR against the inhibitors. We believe that our findings are important for the development of better methods for transgene detection and prevention of false negative results in gene doping testing.     

High-throughput identification of conditionally essential genes in bacteria: from STM to TSM

Signature-tagged mutagenesis (STM) provided the first widely applicable high-throughput method for detecting conditionally essential genes in bacteria by using negative selection to screen large pools of transposon (Tn) mutants. STM requires no prior knowledge of the bacterium's genome sequence, and has been used to study a large number of Gram-positive and Gram-negative species, greatly expanding the repertoires of known virulence factors for these organisms. Originally, hybridization of radiolabelled probes to colony or dot blots was used to detect differences in populations of tagged mutants before and after growth under a selective condition. Modifications of the tag detection method involving polymerase chain reaction (PCR) amplification and visualisation by gel electrophoresis have been developed and can be automated through the use of robotics. Genetic footprinting is another negative selection technique that uses PCR amplification to detect loss of mutants from a pool. Unlike PCR-STM, this technique allows direct amplification of Tn-flanking sequences. However, it requires the bacterium's whole genome sequence in order to design specific primers for every gene of interest. More recently, a number of techniques have been described that combine the negative-selection principle of STM and genetic footprinting with the genome-wide screening power of DNA microarrays. These techniques, although also requiring whole genome sequences, use either a form of linker-mediated or semi-random PCR to amplify and label Tn-flanking regions for hybridization to microarrays. The superior sensitivity microarray detection allows greater numbers of mutants to be screened per pool, as well as determination of the coverage/distribution of insertions in the library prior to screening, two significant advantages over STM.     

肝水解肽样品中牛源及猪源性成分的PCR检测

目的:建立肝水解肽各工艺步骤段中样品的PCR检测方法,用于检测生产原料的动物来源是牛源性或猪源性。方法:分别对猪源性及牛源性肝水解肽各工艺步骤段中样品进行了DNA提取,并加入猪、牛特异性引物进行扩增。对扩增产物进行酶切及测序验证。结果:工艺段为超滤液四之前的样品均可提取并扩增出相应的DNA片段,牛源性PCR产物与Genbank中牛序列的同源性为100%,猪源性PCR产物与猪序列的同源性为99.4%,PCR扩增的检测限为0.5 mg·mL-1的肝细胞酶解液。结论:可用本方法检测肝水解肽各工艺步骤段中超滤液四之前的样品。     

A multiplex qPCR assay for transgenes detection: A novel approach for gene doping control in horseracing using conventional laboratory setup

Illicit administration of transgene into horses is a form of gene doping that has been a key concern in equine sports. The large number of potential performance-enhancing transgenes has demanded a cost-effective and reliable detection method. Multiplex qPCR is a relevant technique, but the cross-talking between fluorophores and high background noise limits the method sensitivity and specificity. This study reports a simpler multiplexing approach by using the same fluorophore for four hydrolysis probes each targeting one of the four transgenes: human growth hormone, insulin-like growth factor 1, equine erythropoietin and interleukin-10. Any positive findings from this multiplex qPCR assay can then be confirmed by individual qPCR assays to identify potential transgene(s). This has effectively eliminated the cross-talking issue and allowed an improved signal-to-noise than conventional multiplex qPCR assay. It has also removed the limitation imposed by the available choice of fluorophores and optical channels of qPCR instruments on the number of transgenes that can be analysed in a multiplex qPCR assay. This novel multiplex qPCR has been successfully validated. The estimated limits of detection were ~1500–2500 copies/mL of blood, thus demonstrating comparable sensitivity with the corresponding duplex qPCR assays. Concurring results were obtained by analysing hundreds of official blood samples provided by racehorses with this multiplex qPCR assay and the accredited individual duplex qPCR assays. This novel multiplex qPCR assay for detecting multiple transgenes is a cost-effective screening method using a conventional laboratory setup and has opened up the potential to include the testing of additional transgenes in a single assay.     

Development of PCR,LAMP and qPCR Assays for the Detection of Aflatoxigenic Strains of Aspergillus flavus and A. parasiticus in Hazelnut

Aspergillus flavus and A. parasiticus are two species able to produce aflatoxins in foodstuffs, and in particular in hazelnuts, at harvest and during postharvest phase. As not all the strains of these species are aflatoxin producers, it is necessary to develop techniques that can detect aflatoxigenic from not aflatoxigenic strains. Two assays, a LAMP (loop-mediated isothermal amplification) and a real time PCR with TaqMan^® probe were designed and validated in terms of specificity, sensitivity, reproducibility, and repeatability. The capability of the strains to produce aflatoxins was measured in vitro and both assays showed to be specific for the aflatoxigenic strains of A. flavus and A. parasiticus. The limit of detection of the LAMP assay was 100–999 picograms of DNA, while the qPCR detected 160 femtograms of DNA in hazelnuts. Both techniques were validated using artificially inoculated hazelnuts and naturally infected hazelnuts. The qPCR was able to detect as few as eight cells of aflatoxigenic Aspergillus in naturally infected hazelnut. The combination of the LAMP assay, which can be performed in less than an hour, as screening method, with the high sensitivity of the qPCR, as confirmation assay, is able to detect aflatoxigenic strains already in field, helping to preserve the food safety of hazelnuts.     

地鼠多瘤病毒PCR检测方法的建立

目的  建立地鼠多瘤病毒（hamster polyomavirus, HaPyV）PCR检测方法,并应用于乙型脑炎减毒活疫苗生产过程中地鼠肾细胞的感染检测。方法  设计针对HaPyV衣壳蛋白VP1基因片段的特异引物,以含HaPyV VP1片段的质粒PMD19T-HaPyV688为模板进行PCR扩增并测序确认扩增产物。以多种病毒DNA为模板进行PCR扩增并对扩增产物进行斑点杂交鉴定以验证PCR方法的特异性。将定量的质粒PMD19T-HaPyV688 10倍系列稀释后进行PCR敏感性检测。以小鼠多瘤病毒(murine polyomavirus, MuPyV)检验PCR法对质粒DNA和病毒DNA检测的一致性。应用建立的方法对生产用地鼠肾细胞悬液进行HaPyV DNA检测。结果  仅含有质粒PMD19T-HaPyV688和MuPyV DNA的样品能够扩增出600 bp的片段,经斑点杂交及测序证明其分别为HaPyV和MuPyV的VP1特异基因片段。PCR所能检出的PMD19T-HaPyV688和MuPyV 的最少DNA拷贝数分别为5300和590。7个亚批生产用地鼠肾细胞悬液的HaPyV检测结果均为阴性。结论  建立的PCR方法具有较高的敏感性和特异性,可用于生产用地鼠肾细胞HaPyV感染的检测。     

蝙蝠蛾拟青霉与冬虫夏草关系的分子系统学研究

以5月和6月两个不同时段采集的分别产于青海省化隆县和四川省康定县的冬虫夏草(Cordyceps sinensis)新鲜子实体为材料，采用分子生物学方法，以rDNA-ITS序列为分子标记，对蝙蝠蛾拟青霉(Paecilomyces hepiali)与冬虫夏草之间的关系进行了探讨。以真菌通用引物ITS1/ITS4分别对蝙蝠蛾拟青霉及冬虫夏草子座和僵虫基因组DNA进行PCR扩增，获得的片段克隆到pMD18-T Vector上进行测序，结果表明，随机挑取的46个克隆与某些已在GenBank中注册的中国被毛孢(Hirsutella sinensis) 或冬虫夏草的rDNA-ITS序列的一致性均在99%以上，但与蝙蝠蛾拟青霉的rDNA-ITS序列的一致性约为72%。根据蝙蝠蛾拟青霉的rDNA-ITS序列设计了两对特异引物，分别以不同产地及不同生长时期共4个批次的冬虫夏草子座和僵虫基因组DNA为模板，进行PCR及巢式PCR扩增，均得到了相应片段，该片段与蝙蝠蛾拟青霉的rDNA-ITS序列具有100%的一致性。在GenBank中注册号为AB067740的另一个标明为冬虫夏草的rDNA-ITS序列与注册号为AJ309353的中国被毛孢的rDNA-ITS序列一致性仅为87.3%，但根据AB067740序列设计特异引物，也从冬虫夏草基因组DNA中扩增到其相应序列。研究结果表明，在天然冬虫夏草中除了中国被毛孢之外，还普遍存在着蝙蝠蛾拟青霉等内寄生菌。     

Low-copy transgene detection using nested digital polymerase chain reaction for gene-doping control

Gene doping is prohibited for fair competition in human and horse sports. One style of gene doping is the administration of an exogeneous gene, called a transgene, to postnatal humans and horses. Although many transgene detection methods based on quantitative polymerase chain reaction (PCR), including real-time PCR and digital PCR, have been recently developed, it remains difficult to reliably detect low-copy transgenes. In this study, we developed and validated a nested digital PCR method to specifically detect low-copy transgenes. The nested digital PCR consists of (1) preamplification using conventional PCR and (2) droplet digital PCR detection using a hydrolysis probe. Using 5, 10, 20, 60 and 120 transgene copies as template, 496.0, 1089.7, 1820.7, 4313.3 and 7840.0 copies per microlitre, respectively, were detected using our nested digital PCR. Although high concentrations of phenol, proteinase K, ethanol, EDTA, heparin and genomic DNA all inhibited preamplification, their effects on the digital PCR detection were limited. Once preamplification was successful, even substitution of bases within the primers and probes had minimal effects on transgene detection. The nested digital PCR developed in this study successfully detected low-copy transgenes and can be used to perform a qualitative test, indicating its usefulness in the prevention of false positives and false negatives in gene-doping detection.     

用PCR法检测致肾盂肾炎大肠杆菌papA基因

目的用PCR方法检测致肾盂肾炎大肠杆菌(UPEC)的papA基因,探索建立UPEC的鉴定方法。方法根据已发表的F     

多形类杆菌的分子生物学鉴定

目的用聚合酶连反应技术(PCR)鉴定多形类杆菌。方法采用16S rRNA基因序列为靶基因设计引物,建立PCR反应体系对该种细菌进行鉴定,并测定PCR产物序列,验证PCR反应的特异性。结果3株多形类杆菌能被扩增,其他菌株和基因组不能被扩增;所测得的PCR产物序列和标准的多形类杆菌16S rRNA基因序列99%同源。结论建立PCR反应体系能特异地鉴定多形类杆菌。     

Towards a robust test to detect gene doping for anabolic enhancement in human athletes

Success in gene therapy in treating human disease makes this technology attractive to enhance athletic performance, creating the need for gene doping detection. In 2021, World Anti-Doping Agency (WADA) approved the first gene doping test. Here, we describe a new method to detect doping with four additional genes, follistatin, growth hormone 1, growth hormone–releasing hormone and insulin-like growth factor 1, that may improve performance by increasing muscle size and strength. The method utilises four hydrolysis probe-based polymerase chain reaction (PCR) assays that target the transgenes based on the coding sequence of the four endogenous genes. The assays are specific, reproducible and capable to detect five copies of transgene in the presence of very similar endogenous gene in 25,000 times excess. To underpin reliable and comparable routine method performance by doping testing laboratories, a synthetic reference material for the method was designed and generated following the ISO Guide 35. The complete method was validated in blood samples using plasma as extraction matrix and QIAamp DNA blood midi DNA extraction kit. All blood samples from different donors (n = 8) simulated to be negative or positive (1500 transgene copies spiked per millilitre of blood) for the transgenes were reported correctly. The new method that targets four additional genes will extend the capabilities of laboratories involved in doping control to protect athletes' health, fairness and equality.     

EPO transgene detection in dried blood spots for antidoping application

The modification of gene expression to treat diseases is a field of research with exponential growth. As doping in sport closely follows emerging therapies, a surveillance of the modification of gene expression to enhance performance is needed. The gene coding for erythropoietin (EPO) is one target of interest. Since 2010, several protocols have been proposed to identify EPO gene doping by focusing on the presence in blood of a transgene that differ in size from the endogenous gene sequence, normally found in the human DNA. In this work, our aim was to validate an easily applicable method for EPO gene doping detection in dried blood spots (DBS). We evaluated the detection of EPO transgene in 20-μl DBS after the spike of a plasmid carrying the EPO transgene in whole blood. Three different DBS were compared: Nucleic-Card™, Whatman® 903, and the volumetric 20-μl VAMS™. Detection was performed with real-time polymerase chain reaction (PCR) and validated with two Taqman assays (one commercial and one custom) specific for the EPO transgene. The initial testing procedure could be done using one assay (custom) and the confirmation using the second one (commercial Taqman) with a final check of the size of the PCR product. Starting from 20-μl dried blood, 1000 copies of EPO transgene could efficiently be detected with the three types of DBS, VAMS showing a slightly better sensitivity. No loss of sensitivity was observed after 1-month storage of DBS at room temperature. This method could be applied to DBS collected during doping controls and allows reanalysis.     

基因扩增法检测军团杆菌DNA的研究

本研究采用聚合酶链式反应（PCR），建立检测军团菌DNA的实验诊断方法。结果显示：采用军团菌属5SrRNA编码区特异性引物进行扩增，11种军团菌（包括8种嗜肺军团菌）均可见104bp的阳性扩增带；采用嗜肺军团菌巨噬细胞感染增强（mip）基因编码区特异性引物扩增，所有8种嗜肺军团菌均可见650bp的特异性扩增带，所有3种非嗜肺军团菌扩增结果为阴性。检测敏感性为60fg左右。该研究技术的建立，为军团病的早期诊断和环境监测及控制爆发流行提供了一种快速、敏感、特异的新方法。     

Simultaneous Detection of Ricin and Abrin DNA by Real-Time PCR (qPCR)

Ricin and abrin are two of the most potent plant toxins known and may be easily obtained in high yield from the seeds using rather simple technology. As a result, both toxins are potent and available toxins for criminal or terrorist acts. However, as the production of highly purified ricin or abrin requires sophisticated equipment and knowledge, it may be more likely that crude extracts would be used by non-governmental perpetrators. Remaining plant-specific nucleic acids in these extracts allow the application of a real-time PCR (qPCR) assay for the detection and identification of abrin or ricin genomic material. Therefore, we have developed a duplex real-time PCR assays for simultaneous detection of ricin and abrin DNA based on the OmniMix HS bead PCR reagent mixture. Novel primers and hybridization probes were designed for detection on a SmartCycler instrument by using 5′-nuclease technology. The assay was thoroughly optimized and validated in terms of analytical sensitivity. Evaluation of the assay sensitivity by probit analysis demonstrated a 95% probability of detection at 3 genomes per reaction for ricin DNA and 1.2 genomes per reaction for abrin DNA. The suitability of the assays was exemplified by detection of ricin and abrin contaminations in a food matrix.     

Patent Evaluation: A Method for the Detection of Point Mutations

Summary

Novelty: A novel method for the detection of point mutations and genetic variations is described. The method is based on primer extension and the incorporation of detectable nucleoside triphosphates.

Biology: The method for detection of point mutations in sequence variations is dependent on the knowledge of the sequence adjacent to the variable region, preferably immediately toward the 3′ end of the variable nucleotide. By selecting the detection step primers from the region immediately adjacent to the variable nucleotide, this variation can be detected after incorporation of as few as one nucleotide triphosphate. Labelled nucleotide triphosphates matching the variable nucleotide are added and the incorporation of label into the detection step primer is measured. This method, in conjunction with an amplification method such as PCR, can be used with a wide variety of radioactive and non-radioactive detection methods.     

用多重PCR方法鉴别不同卡介苗菌株

目的用多重PCR方法鉴别不同卡介苗菌株。方法根据卡介苗DNA的6个目的区域的特点设计引物,进行多重PCR扩增,然后电泳检测分析。结果多重PCR方法能够鉴别不同卡介苗亚株。结论应用多重PCR方法鉴别卡介苗菌株具有专一性好、重复性高、快速简便等优点。     

New application of the CRISPR-Cas9 system for site-specific exogenous gene doping analysis

The increased potential for gene doping since the introduction of gene therapy presents the need to develop antidoping assays. We therefore aimed to develop a quick and simple method for the detection of specifically targeted exogenous doping genes utilizing an in vitro clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9) system. A human erythropoietin (hEPO) is a drug frequently used for doping in athletes, and gene doping using gene transfer techniques may be attempted. Therefore, we selected hEPO gene as a model of exogenous doping gene, and complemental single guide RNA (sgRNA) was designed to specifically bind to the four exon–exon junctions in the hEPO cDNA. For the rapid reaction of CRISPR-Cas9, further optimization was performed using an open-source program (CRISPOR) that avoids TT and GCC motifs before the protospacer adjacent motif (PAM) domain and predicts the efficiency of the sgRNA. We optimized the in vitro Cas9 assay and dual use of sgRNA for double cleavage and identified the limit of detection (LOD) of the 1.25 nM of the double cleavage method. We expect that the improved CRISPR-Cas9 method can be used for antidoping analysis of gene doping.