Quantification of donor microchimerism in sex-mismatched porcine allotransplantation by competitive PCR

The persistence of donor cells in recipient circulation and peripheral tissues post-transplantation has been demonstrated in solid organ allotransplantation and xenotransplantation models. Although this state of microchimerism has been postulated as the basis for graft acceptance, chimerism has not been directly linked to the maintenance of peripheral tolerance or prevention of rejection. Studies have demonstrated that the qualitative presence or absence of donor microchimerism bears no association with graft acceptance. Our preliminary work suggests that there is a threshold chimerism necessary for the induction of donor-specific hyporesponsiveness. Because the kinetics of donor cell accumulation and distribution in allograft recipients are largely unknown, quantitative analyses are needed to evaluate chimerism's significance to donor-specific tolerance. We developed a quantitative, competitive PCR assay to precisely measure the amount of chimerism in male to female transplant pairs by targeting the sex-determining region of the Y chromosome (SRY gene). Traditionally, this technique requires that serial known amounts of an SRY-specific competitive template (CT) be coamplified with a constant amount of sample DNA to determine the equivalence point of the relative band intensities of the PCR products. However running a panel of PCRs with CT amounts above and below the equivalence point to generate a standard curve for ever' sample is laborious. Here we describe the generation of a single standard curve that permits the rapid and reliable quantification of microchimerism after coamplification of sample DNA with a single amount of CT.     

Applications of quantitative PCR in the biosafety and genetic stability assessment of biotechnology products

High throughput screening, increased accuracy and the coupling of real-time quantitative PCR (Q-PCR) to robotic set-up systems are beginning to revolutionise biotechnology. Applications of Q-PCR within biotechnology are discussed with particular emphasis on the following areas of biosafety and genetic stability testing: (a) determination of the biodistribution of gene therapy vectors in animals; (b) quantification of the residual DNA in final product therapeutics; (c) detection of viral and bacterial nucleic acid in contaminated cell banks and final products; (d) quantification of the level of virus removal in process validation viral clearance studies; (e) specific detection of retroviral RT activity in vaccines with high sensitivity; and (f) transgene copy number determination for monitoring genetic stability during production. Methods employed for Q-PCR assay validation as required in ICH Topic Q2A Validation of Analytical Methods: Definitions and Terminology (1st June 1995) are also reviewed.     

Comparison of methods for quantification of cytochrome cd(1)-denitrifying bacteria in environmental marine samples

Two PCR primer sets were developed for the detection and quantification of cytochrome cd(1)-denitrifying bacteria in environmental marine samples. The specificity and sensitivity of these primers were tested. Both primer sets were suitable for detection, but only one set, cd3F-cd4R, was suitable for the quantification and enumeration of the functional community using most-probable-number PCR and competitive PCR techniques. Quantification of cytochrome cd(1) denitrifiers taken from marine sediment and water samples was achieved using two different molecular techniques which target the nirS gene, and the results were compared to those obtained by using the classical cultivation method. Enumerations using both molecular techniques yielded similar results in seawater and sediment samples. However, both molecular techniques showed 1,000 or 10 times more cytochrome cd(1) denitrifiers in the sediment or water samples, respectively, than were found by use of the conventional cultivation method for counting.     

Internally controlled real-time PCR method for quantitative species-specific detection and vapA genotyping of Rhodococcus equi

We developed a novel quantitative real-time PCR (Q-PCR) method for the soil actinomycete Rhodococcus equi, an important horse pathogen and emerging human pathogen. Species-specific quantification was achieved by targeting the chromosomal monocopy gene choE, universally conserved in R. equi. The choE Q-PCR included an internal amplification control (IAC) for identification of false negatives. A second Q-PCR targeted the virulence plasmid gene vapA, carried by most horse isolates but infrequently found in isolates from other sources. The choE-IAC and vapA assays were 100% sensitive and specific as determined using 178 R. equi isolates, 77 nontarget bacteria, and a panel of 60 R. equi isolates with known vapA+ and vapA-negative (including vapB+) plasmid genotypes. The vapA+ frequency among isolate types was as follows: horse, 85%; human, 20%; bovine and pig, 0%; others, 27%. The choE-IAC Q-PCR could detect up to one genome equivalent using R. equi DNA or 100 bacteria/ml using DNA extracted from artificially contaminated horse bronchoalveolar lavage (BAL) fluid. Quantification was linear over a 6-log dynamic range down to approximately 10 target molecules (or 1,000 CFU/ml BAL fluid) with PCR efficiency E of >0.94. The vapA assay had similar performance but appeared unsuitable for accurate (vapA+) R. equi quantification due to variability in target gene or plasmid copy number (1 to 9). The dual-reaction Q-PCR system here reported offers a useful tool to both medical and veterinary diagnostic laboratories for the quantitative detection of R. equi and (optional) vapA+ "horse-pathogenic" genotype determination.     

Human DNA polymorphisms and methods of analysis.

The current predominant method of analyzing base substitution polymorphisms, RFLP analysis, is likely to be gradually supplanted by methods based on PCR because of the improved sensitivity and genotyping rate. The most promising PCR methods for analysis appear to be allele-specific PCR and single-stranded conformational analysis. The single-stranded conformation approach has already been applied to the scanning of cystic fibrosis exons for new mutations. Linkage mapping projects that cover large segments of the human genome will probably rely, in the coming years, primarily on tandem repeat polymorphisms, particularly microsatellite polymorphisms. Microsatellite polymorphisms have at least a fourfold advantage over base substitution RFLPs because they are twice as informative and can be typed at at least twice the rate. The facioscapulohumeral muscular dystrophy gene was recently mapped in just 6 weeks using microsatellite polymorphisms. Because of the informativeness handicap, it will be difficult for base substitution polymorphisms to overtake tandem repeat markers for large-scale linkage mapping. Methods that allow base substitution polymorphisms to be typed at two or three times the rate of microsatellite markers would have to be developed. Most of the other applications of DNA polymorphisms described in the introduction are also increasingly likely to rely on highly informative tandem repeat markers in the future. Methods for analysis will probably be based on PCR. It is easy to envisage, for example, an automated method for large-scale DNA fingerprinting of individuals based upon a standard set of highly informative, dependable microsatellite polymorphisms. Methods for analyzing base substitution polymorphisms will continue to be important for the diagnostic detection of disease-gene alleles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microfluidic chip-based assay for post-hematopoietic stem cell transplantation chimerism monitoring using polymorphic tandem repeat markers

Following hematopoietic stem cell transplantation (HSCT), it is important to determine whether engraftment is successful and to track the dynamic changes of the graft. Tandem repeats such as minisatellites and microsatellites are currently the most established markers for chimerism application. We have developed a reliable method to quantitatively evaluate engraftment status in post-allogeneic HSCT patients using variable number of tandem repeat (VNTR) markers and “lab-on-a-chip” microfluidic electrophoresis technology. Following identification of an informative marker by conventional polymerase chain reaction (PCR), donor chimerism percentage was calculated based on a standard curve generated from artificially mixed patient-donor DNA-specific alleles in serial dilutions. All PCR products were mixed with commercial gel dye and loaded into Agilent DNA 1000 microfluidic LabChips for DNA sizing and quantitation. In 44 patients, separation of pretransplant and donor DNA fragments was resolved clearly and accomplished rapidly within 30 min. Chimerism analysis using this platform is able to detect an amount as low as 6.3% donor DNA with acceptable coefficient of variation values. We also demonstrated concordant chimerism analysis findings using both microchip tandem repeats and real-time PCR quantitation of insertion-deletion polymorphisms. This microchip platform obviates the need for fluorescently labeled primers or any post-PCR sample manipulation. Quantitative monitoring of post-HSCT chimerism status using microfluidic electrophoresis is a useful tool for both large- and small-scale post-HSCT chimerism centers.     

Enumeration of Archaea and Bacteria in seafloor basalt using real-time quantitative PCR and fluorescence microscopy

A SYBR Green real-time quantitative PCR (Q-PCR) assay for the detection and quantification of Bacteria and Archaea present in the glassy rind of seafloor basalts of different ages and water depths is presented. Two sets of domain-specific primers were designed and validated for specific detection and quantification of bacterial and archaeal 16S rRNA genes in DNA extracted from basaltic glass. Total cell numbers were also estimated by fluorescence microscopy analysis of SYBR Gold-stained samples. The results from the two different approaches were concurrent, and Q-PCR results showed that the total number of cells present in basalts was in the range from 6 x 10(5) to 4 x 10(6) cells g(-1) basaltic glass. Further, it was demonstrated that these cells were almost exclusively from the domain Bacteria. When applying the same methods on samples of different ages (22 years-0.1 Ma) and water depths (139-3390 mbsl), no significant differences in cell concentrations or in the relative abundance of Archaea and Bacteria were detected.     

人博卡病毒TaqMan探针实时定量PCR检测方法的建立及初步应用

目的:建立人博卡病毒(HBoV)核酸特异、快速、敏感的TaqMan探针实时定量PCR检测方法,并对临床样本进行检测。方法:比对编码HBoV非结构蛋白NP-1的基因序列,选取其保守片段设计引物和探针,建立实时荧光定量PCR检测方法,并与传统PCR方法进行比较,然后分别对两者的灵敏性、特异性、稳定性及临床样本检验的适用性等进行评价。结果:所建立的实时定量PCR检测方法可用于HBoV的特异性检测;相对于传统PCR所达到的250拷贝/反应的检测灵敏度,实时定量PCR的检测灵敏度可高达10拷贝/反应,检测范围为109～101拷贝/反应,且具有良好的特异性和重复性;初步用于76份临床呼吸道标本检测,检出阳性5例,高于普通PCR方法(3/76)。结论:建立了HBoV TaqMan探针实时定量PCR检测方法,并可用于临床鼻咽拭子样本的检测,为开展HBoV流行病学监测及早期临床诊断提供了技术手段。     

Molecular characterization of microbial populations at two sites with differing reductive dechlorination abilities

This study compares three molecular techniques, including terminal restriction fragment length polymorphism (T-RFLP), RFLP analysis with clone sequencing, and quantitative PCR (Q-PCR) for surveying differences in microbial communities at two contaminated field sites that exhibit dissimilar chlorinated solvent degradation activities. At the Idaho National Engineering and Environmental Laboratory (INEEL), trichloroethene (TCE) was completely converted to ethene during biostimulation with lactate. At Seal Beach, California, perchloroethene (PCE) was degraded only to cis-dichloroethene (cDCE) during biostimulation but was degraded to ethene after bioaugmentation with a dechlorinating culture containing Dehalococcoides strains. T-RFLP analysis showed that microbial community composition differed significantly between the two sites, but was similar within each site among wells that had low or no electron donor exposure. Analysis of INEEL clone libraries by RFLP with clone sequencing revealed a complex microbial population but did not identify any Dehalococcoides strains. Q-PCR targeting the 16S rRNA gene of Dehalococcoides strains – known for their unique capability to dechlorinate solvents completely to ethene – revealed a significant population at INEEL, but no detectable population at Seal Beach prior to bioaugmentation. Detection of Dehalococcoides by Q-PCR correlated with observed dechlorination activity and ethene production at both sites. Q-PCR showed that Dehalococcoides was present in even the pristine well at INEEL, suggesting that the difference in dechlorination ability at the two sites was due to the initial absence of this genus at Seal Beach. Of the techniques tested, Q-PCR quantification of specific dechlorinating species provided the most effective and direct prediction of community dechlorinating potential.     

一种简便经济的HLA-DRB1*15基因检测方法的建立

目的:建立一种简便经济的HLA-DRB1*15基因(血清学特异性为DR15)PCR扩增方法,并与进口试剂盒进行比较。方法:以201例准备进行骨髓移植的供患者为研究对象,用我们设计的PCR扩增条件,即HLA-DRB1*15基因和其关联基因HLA-DRB5*(血清学特异性为DR51)分别与内参照基因同管扩增,判定的结果与HLA-DR进口试剂盒的分型结果进行比较。结果:在这201例供患者中,判定为DRB1*15阳性的有85例,判定为DRB1*15阴性的有116例,在判定为DRB1*15阴性的116例中发现有5例为DRB1*16阳性。与HLA-DR基因分型检测结果进行比较,结果显示采用我们设计的DRB1*15扩增方法判定的结果与HLA-DR基因分型检测结果完全一致。结论:我们建立的DRB1*15低分辨扩增方法结果正确,是一种简便经济的方法,值得在临床实验室推广。     

Development of quantitative real-time PCR assays for detection and quantification of surrogate biological warfare agents in building debris and leachate

Evaluation of the fate and transport of biological warfare (BW) agents in landfills requires the development of specific and sensitive detection assays. The objective of the current study was to develop and validate SYBR green quantitative real-time PCR (Q-PCR) assays for the specific detection and quantification of surrogate BW agents in synthetic building debris (SBD) and leachate. Bacillus atrophaeus (vegetative cells and spores) and Serratia marcescens were used as surrogates for Bacillus anthracis (anthrax) and Yersinia pestis (plague), respectively. The targets for SYBR green Q-PCR assays were the 16S-23S rRNA intergenic transcribed spacer (ITS) region and recA gene for B. atrophaeus and the gyrB, wzm, and recA genes for S. marcescens. All assays showed high specificity when tested against 5 ng of closely related Bacillus and Serratia nontarget DNA from 21 organisms. Several spore lysis methods that include a combination of one or more of freeze-thaw cycles, chemical lysis, hot detergent treatment, bead beat homogenization, and sonication were evaluated. All methods tested showed similar threshold cycle values. The limit of detection of the developed Q-PCR assays was determined using DNA extracted from a pure bacterial culture and DNA extracted from sterile water, leachate, and SBD samples spiked with increasing quantities of surrogates. The limit of detection for B. atrophaeus genomic DNA using the ITS and B. atrophaeus recA Q-PCR assays was 7.5 fg per PCR. The limits of detection of S. marcescens genomic DNA using the gyrB, wzm, and S. marcescens recA Q-PCR assays were 7.5 fg, 75 fg, and 7.5 fg per PCR, respectively. Quantification of B. atrophaeus vegetative cells and spores was linear (R(2) > 0.98) over a 7-log-unit dynamic range down to 10(1) B. atrophaeus cells or spores. Quantification of S. marcescens (R(2) > 0.98) was linear over a 6-log-unit dynamic range down to 10(2) S. marcescens cells. The developed Q-PCR assays are highly specific and sensitive and can be used for monitoring the fate and transport of the BW surrogates B. atrophaeus and S. marcescens in building debris and leachate.     

Relationship between chimerism and tolerance in a kidney transplantation model

The persistence of donor leukocytes in recipients of organ allografts has been associated with long-term graft acceptance. However, it remains unclear whether this peripheral donor cell microchimerism plays an active role in graft acceptance or is simply a consequence of the maintenance of sufficient immunosuppression to avoid rejection. A model of kidney transplantation between swine leukocyte Ag (SLA)-matched miniature swine, in which tolerance can be established with or without immunosuppressive treatment, has been used to study the correlation between donor leukocyte chimerism and kidney graft acceptance. SLA-identical kidney transplants were performed from animals positive for an allelic pig leukocyte Ag to animals negative for this marker. SLA-identical kidney transplant recipients given a 12-day course of cyclosporine (CyA) (n = 3) became tolerant, showing stable serum creatinine levels (1-2 mg/dl) after cessation of CyA treatment. Donor cell chimerism (0.2-0.7%) was present by FACS in all three animals with peak levels detected at 3 wk. Two control animals receiving SLA-identical kidney grafts without CyA also showed stable serum creatinine levels and became tolerant. However, in neither of these animals could donor leukocytes be detected in the peripheral blood beyond 1 wk following transplantation. In one additional control animal, ureteral obstruction occurred at day 10, and was associated with additional peripheral chimerism, presumably related to inflammation rather than to immune status. These results indicate that the persistence of donor cell chimerism is not a requirement for the maintenance of tolerance to organ allografts in this model.     

DNA分子标记在实验动物遗传分析中的应用

DNA分子标记技术很多,基本都是建立在RFLP、PCR和重复顺序的基础上的。本文重点介绍了限制性片段长度多态性（RFLP）标记、随机扩增多态性DNA（RAPD）标记、微卫星DNA（STR）标记、DNA指纹（DFP）标记、扩增片段长度多态性（AFLP）标记等几种重要的DNA分子标记技术的定义、结构、分布、组成、保守性、优点及丰富的多态性等。并重点介绍了微卫星DNA（STR）标记在分子遗传监测、遗传多样性分析和遗传血缘关系及个体识别等领域的应用。     

Long-term follow-up of porcine male germ cells transplanted into mouse testes

This study investigated the effect of increased phylogenetic distance on the outcome of spermatogonial transplantation, with porcine donors and mice recipients. It was designed to develop a technique for detecting foreign donor cells in recipient animals. Porcine male germ cells were harvested from postnatal male testes and incubated with the lipophilic membrane dye PKH-26. For transplantation, approximately 10(6) PKH-26-labelled porcine male germ cells were injected into the efferent ducts of mouse testes. Animals were sacrificed at post-graft days 1, 10, 30, 45, 60 and 150 (n = 5 each). Serial frozen sections of explanted testes were prepared for detecting labelled cells. Transplanted porcine donor cells were easily detected in the recipient tubules for 8 weeks. After transplantation, we could detect both incorporation into the basement membrane and differentiation of grafted porcine donor cells by our double detection system, using PKH staining and slide PCR. However, our RT-PCR and apoptosis results revealed that most of the grafted porcine male donor cells could not differentiate past early-meiotic spermatocytes. We could induce partial differentiation of xenografted porcine donor cells in mouse testes, but not full induction of spermatogenesis. We have developed a very reliable technique for detecting foreign donor cells in recipient animals using a combination of PKH staining and slide PCR methods. Our results provide a valuable experimental model for applying and evaluating this technology in other species.     

Immuno-qPCR detection of the tandem affinity purification (TAP)-tag as a sensitive and accurate tool suitable for large-scale protein quantification

The tandem affinity purification (TAP)-tag has rapidly gained a wide popularity, mostly in studies on protein interactions, but lately also in large-scale protein quantification studies. We have developed an immuno-quantitative real-time PCR (qPCR) method to achieve rapid, sensitive and accurate quantification of TAP-tagged (and protein A-tagged) proteins in yeast with a detection range between 10(7) and 10(10) molecules. The immuno-qPCR protein quantification showed an excellent correlation to the published in vivo fluorescent protein (GFP)-based large-scale protein quantifications, but allowed for a much higher sensitivity. The correlation with published data from the large-scale Western blotting-based quantification of the TAP-tag was lower, but the sensitivity of detection was on roughly the same level. The practical use of the immuno-qPCR approach was demonstrated by analysis of osmo-regulated proteins, where the 2000-fold increase in expression of Catalase (Ctt1p), from an extremely low basal expression, could be accurately quantified. All steps of the method, from cell growth, to protein extraction and determination and the immuno-qPCR reaction itself are potentially amenable to automatization. Therefore, since the TAP-tag and protein A are useful in most model organisms, the immuno-qPCR method is both generic and suitable for large-scale studies.     

Detection of Heterobasidion annosum s. l. [(Fr.) Bref.] in Norway Spruce by Polymerase Chain Reaction

Internal transcribed spacer (ITS) sequences of the rDNA repeat unit of Heterobasidion annosum were used to design specific primers for the detection and quantification of this important forest pathogen by polymerase chain reaction (PCR). Specificity of detection was cross‐checked against a variety of other fungi (saprophytes, root pathogens, mycorrhizal fungi) which may occur in the same environment. As little as 1 pg fungal DNA (equiv. to 10–40 genomes) could be detected in 200 ng spruce root DNA (from 1 mg fresh spruce root). The Heterobasidion‐specific primers allowed simultaneous detection of Armillaria spp. in multiplex PCR. The method was successfully applied to increment cores of Norway spruce from the forest region Tharandter Wald (Saxonia, Germany), Oberbärenburg (East Ore Mountains, Saxonia) and Oberschleissheim (north of Munich, Bavaria).     

Heterogeneous Distribution of Fetal Microchimerism in Local Breast Cancer Environment

Fetal cells enter maternal circulation during pregnancy and persist in the woman’s body for decades, achieving a form of physiological microchimerism. These cells were also evidenced in tumors. We investigated the frequency and concentration of fetal microchimerism in the local breast cancer environment. From 19 patients with confirmed breast neoplasia, after breast surgical resection, we collected three fresh specimens from the tumor core, breast tissue at tumor periphery, and adjacent normal breast tissue. The presence of male DNA was analyzed with a quantitative PCR assay for the sex determining region gene (SRY) gene. In the group of women who had given birth to at least one son, we detected fetal microchimerism in 100% of samples from tumors and their periphery and in 64% (9 of 14) of those from normal breast tissue. The tissues from the tumor and its periphery carry a significantly increased number of SRY copies compared to its neighboring common breast tissue (p = 0.005). The median of the normalized SRY-signal was about 77 (range, 3.2–21467) and 14-fold (range, 1.3–2690) greater in the tumor and respectively in the periphery than in the normal breast tissue. In addition, the relative expression of the SRY gene had a median 5.5 times larger in the tumor than in its periphery (range, 1.1–389.4). We found a heterogeneous distribution of fetal microchimerism in breast cancer environment. In women with sons, breast neoplasia harbors male cells at significantly higher levels than in peripheral and normal breast tissue.     

Absence of replication of porcine endogenous retrovirus and porcine lymphotropic herpesvirus type 1 with prolonged pig cell microchimerism after pig-to-baboon xenotransplantation

Porcine endogenous retrovirus (PERV), porcine cytomegalovirus (PCMV), and porcine lymphotropic herpesvirus (PLHV) are common porcine viruses that may be activated with immunosuppression for xenotransplantation. Studies of viral replication or transmission are possible due to prolonged survival of xenografts in baboon recipients from human decay-accelerating factor transgenic or alpha-1,3-galactosyltransferase gene knockout miniature swine. Ten baboons underwent xenotransplantation with transgenic pig organs. Graft survival was 32 to 179 days. Recipient serial samples of peripheral blood mononuclear cells (PBMC) and plasma were analyzed for PCMV, PERV, and PLHV-1 nucleic acids and viral replication using quantitative PCR assays. The PBMC contained PERV proviral DNA in 10 animals, PLHV-1 DNA in 6, and PCMV in 2. PERV RNA was not detected in any PBMC or serum samples. Plasma PLHV-1 DNA was detected in one animal. Pig cell microchimerism (pig major histocompatibility complex class I and pig mitochondrial cytochrome c oxidase subunit II sequences) was present in all recipients with detectable PERV or PLHV-1 (85.5%). Productive infection of PERV or PLHV-1 could not be demonstrated. The PLHV-1 viral load did not increase in serum over time, despite prolonged graft survival and pig cell microchimerism. There was no association of viral loads with the nature of exogenous immune suppression. In conclusion, PERV provirus and PLHV-1 DNA were detected in baboons following porcine xenotransplantation. Viral detection appeared to be due to persistent pig cell microchimerism. There was no evidence of productive infection in recipient baboons for up to 6 months of xenograft function.     

Microsatellite-based parentage control in cattle

As a new approach to parentage control we developed two multiplex coamplification polymerase chain reaction (PCR) systems containing a total of six different short tandem repeat (STR) loci; the microsatellite polymorphisms were visualized by automated fluorescence detection on the Applied Biosystems 373 DNA Sequencer with 672 Genescan Analysis software. Allele frequency data were determined from 238 animals. Thirty-five bovine parentage control cases not solvable by conventional blood typing could be solved.