Quantitative real-time PCR with automated sample preparation for diagnosis and monitoring of cytomegalovirus infection in bone marrow transplant patients

BACKGROUND: In bone marrow and stem cell transplant patients, the widespread use of preemptive cytomegalovirus (CMV) antiviral therapy necessitates faster, more precise, and more sensitive quantitative laboratory methods for serial viral load monitoring. METHODS: We developed a novel CMV viral load assay using real-time PCR of plasma DNA prepared by an automated robotic workstation. Fluorescent hybridization probes directed at the glycoprotein B (gB) gene (or EcoRI D region) of CMV were used to detect and quantify PCR products. The beta-globin gene was amplified in parallel to control for the efficiency of the extraction and PCR steps. RESULTS: The assay was linear (R = 0.999) from a lower detection limit of 125 copies/mL to 5 x 10(9) copies/mL with a PCR efficiency of 1.975 (gB) or 2.02 (EcoRI D). The viral loads determined by PCRs directed at these two different viral targets were no different (n = 53; R = 0.928). The interassay CV was 3.5%, and the intraassay CV was 1-4%. Compared with a commercially available quantitative competitive PCR assay (Roche MONITOR; R = 0.59), the mean CMV viral load by real-time PCR was 3.1 times higher (mean ratio; P = 0.002). The diagnostic sensitivity and specificity of the real-time assay were 96% and 100%, respectively (n = 147), compared with 74% and 98% for a qualitative PCR assay (Roche AMPLICOR). On a subset of samples, the diagnostic sensitivity of viral culture was no greater than 50% (n = 44). Of 1115 clinical referral samples from 252 patients, 10% of the samples and 18% of the patients had low-level CMV viremia (median, 500 copies/mL). In this predominantly (85%) bone marrow transplant testing cohort, serial CMV viral load results were the predominant clinical trigger for the initiation, monitoring, and cessation of preemptive antiviral therapy. CONCLUSIONS: The combination of automated DNA preparation and semiautomated real-time fluorescent PCR detection allows for a sensitive, precise, and accurate high-throughput assay of CMV viral load that can be used as the laboratory trigger for preemptive antiviral therapy.     

Automated processing of whole blood samples for the determination of immunosuppressants by liquid chromatography tandem-mass spectrometry.

BACKGROUND: Liquid chromatography tandem-mass spectrometry (LC-MS/MS) is an efficient technology for routine determination of immunosuppressants in whole blood; however, time-consuming manual sample preparation remains a significant limitation of this technique. METHODS: Using a commercially available robotic pipetting system (Tecan Freedom EVO), we developed an automated sample-preparation protocol for quantification of tacrolimus in whole blood by LC-MS/MS. Barcode reading, sample resuspension, transfer of whole blood aliquots into a deep-well plate, addition of internal standard solution, mixing, and protein precipitation by addition of an organic solvent is performed by the robotic system. After centrifugation of the plate, the deproteinized supernatants are submitted to on-line solid phase extraction, using column switching prior to LC-MS/MS analysis. The only manual actions within the entire process are decapping of the tubes, and transfer of the deep-well plate from the robotic system to a centrifuge and finally to the HPLC autosampler. Whole blood pools were used to assess the reproducibility of the entire analytical system for measuring tacrolimus concentrations. RESULTS: A total coefficient of variation of 1.7% was found for the entire automated analytical process (n=40; mean tacrolimus concentration, 5.3 microg/L). Close agreement between tacrolimus results obtained after manual and automated sample preparation was observed. CONCLUSIONS: The analytical system described here, comprising automated protein precipitation, on-line solid phase extraction and LC-MS/MS analysis, is convenient and precise, and minimizes hands-on time and the risk of mistakes in the quantification of whole blood immunosuppressant concentrations compared to conventional methods.     

Comparison of volumetric capillary cytometry with standard flow cytometry for routine enumeration of CD34+ cells

BACKGROUND: This study assesses the feasibility of a new volumetric cytometry system for the enumeration of CD34+ cells in apheresis components, peripheral blood, and cord blood samples in routine laboratory work. This system is compared with the following flow cytometry protocols: Milan, ISHAGE, ISHAGE with 7-AAD, and flow-count fluorospheres. STUDY DESIGN AND METHODS: Correlation, linearity, and reproducibility studies were performed for the various methods. Clonogenic cultures were performed, as an external control, to assess the correlation between the number of CD34+ cells per microL and the number of colony-forming units per microL. RESULTS: The linear regression analysis demonstrated that the five methods were comparable (R2 ranged from 0.86 to 0.96 and slopes were close to 1). The CD34+ assay and the flow-count methods showed poor linearity for CD34+ cell counts below 10 cells per microL (R2 = 0.46 and 0.47). The reproducibility assay for a CD34+ count of 10 cells per microL showed a CV of 12 percent and 25 percent for the Milan and CD34+ assay methods, respectively. The mean CV among all five methods for the 46 evaluated samples was 20 percent. There was a strong correlation between the number of CD34+ cells per microL and colony-forming units per microL in cord blood and apheresis samples (r = 0.71-0.81). CONCLUSION: The CD34+ assay is useful in CD34 enumeration in cord blood, leukapheresis samples, and peripheral blood samples and provides comparable results to the Milan, ISHAGE, ISHAGE with 7-AAD, and flow-count methods. Nevertheless, peripheral blood samples with low CD34 absolute counts (below 10 cells/microL) should be analyzed by alternative flow cytometry protocols. Even though the same operator performed the study in a single laboratory, the high inter-method CV suggests that differences in sample preparation and gating strategy are factors that increase variability. Protocols with fewer intermediate steps or fully automated protocols such as the CD34+ assay are expected to reduced intra- and inter-laboratory variability.     

Detection of diarrhoeal pathogens in human faeces using an automated, robotic platform

Infectious diarrhoeal diseases represent a major socio-economic burden to humans, and are linked to a range of pathogens, including viruses, bacteria and protists. The accurate detection of such pathogens is central to control. However, detection often relies on methods that have limited diagnostic sensitivity and specificity. Here, we assessed an automated, robotic platform for the simultaneous detection of eight major pathogens associated with infectious diarrhoea. Genomic DNA samples (n = 167) from faeces from humans with diarrhoea and diagnosed as cryptosporidiosis, and 100 uninfected control subjects, were tested for adenovirus 40/41, norovirus, Clostridium difficile, Campylobacter, Salmonella, Shigella, Cryptosporidium and Giardia by multiplexed-tandem PCR, and also characterized by single-strand conformation polymorphism analysis (SSCP) and selective sequencing. All 167 samples tested positive for Cryptosporidium, five for adenovirus 40/41, four for Campylobacter, three for C. difficile and seven for Shigella spp., with no false positive results for any assay. The automated PCR exhibited a high sensitivity, with <10 individual pathogens being readily detected. The robotic detection platform assessed here represents a sensitive, high-throughput tool for key pathogens linked to infectious diarrhoea in humans. This platform requires little molecular biological expertise and is well suited to various diagnostic facilities and settings.     

Determination of 24 minor red blood cell antigens for more than 2000 blood donors by high-throughput DNA analysis

BACKGROUND: A "BeadChip" array permits reliable simultaneous DNA typing of single-nucleotide polymorphisms for minor blood groups. A high-throughput DNA analysis was studied as a routine method of phenotype prediction and software was developed to interpret and analyze the large volume of data points. STUDY DESIGN AND METHODS: DNA was extracted from whole blood of donors of known phenotypes and self-identified ethnicity. Analysis of single-nucleotide polymorphisms (SNPs) associated with 24 antigens of 10 blood group systems was performed with BeadChips (BioArray Solutions), and the results were compared to historical serologic typings. Phenotypes were predicted for individual samples, and phenotype prevalence was determined for ethnicities. The BeadChip was expanded to incorporate SNPs that silence the S antigen, validated, and tested with 369 DNA samples. A time-motion analysis was conducted. RESULTS: Results of BeadChip analyses were concordant with prediction of antigen negativity for 4,510 antigens. Eight discordant results were due to silencing of GYPB(S) and 16 were likely errors in recording serological results or data entry. The analyses produced 19,457 antigen-negative typings not serologically defined, identified 21 rare donors (Co(a-b+) [n = 1], Jo(a-) [n = 6], S-s-[n = 12], and K+k-[n = 2]), and determined allele frequencies and antigen prevalence for four ethnicities. The expanded panel detected 30 SS, 235 ss, 100 Ss, and 4 U- samples. The format processes 192 DNA samples (two plates) per 8-hour shift per technician, including automated data analysis and report generation. CONCLUSION: DNA analysis with BeadChip format, combined with computerized data entry and analysis, permits the prediction of minor blood group antigens.     

Rapid detection of codon 460 mutations in the UL97 gene of ganciclovir-resistant cytomegalovirus clinical isolates by real-time PCR using molecular beacons

A rapid real-time polymerase chain reaction (PCR) assay using molecular beacons has been developed for the simultaneous detection of wild-type and mutant strains of cytomegaloviruses (CMV) with respect to codon 460 of the UL97 gene has been developed. The molecular beacons were designed to complement the wild-type codon 460 or the mutant sequence arising from a single base-pair difference (point mutation). Discrimination between wild-type and mutant templates was demonstrated as the beacons did not generate fluorescence with their respective mismatch targets but only with those that they were designed to perfectly anneal with. Samples that harbor mixed populations of CMV could also be readily recognized. Applied to a small number of clinical samples, the retrospective screening by this assay are in general concordance with that obtained by PCR-RFLP. Using molecular beacons strategy, codon 460 mutation was detected in ten out o the total number of 40 samples, whereas the latter method identified nine samples as containing the mutation. The discrepant result arose from the genotyping of one clinical sample as mixed (containing both wild-type and mutant CMV strains) by molecular beacons but as wild-type by PCR-RFLP, suggesting that this real-time strategy is possibly more sensitive for mutation analysis.     

Comparative evaluation of automated and manual commercial DNA extraction methods for detection of Francisella tularensis DNA from suspensions and spiked swabs by real-time polymerase chain reaction

This study evaluated commercial automated and manual DNA extraction methods for the isolation of Francisella tularensis DNA suitable for real-time polymerase chain reaction (PCR) analysis from cell suspensions and spiked cotton, foam, and polyester swabs. Two automated methods, the MagNA Pure Compact and the QIAcube, were compared to 4 manual methods, the IT 1-2-3 DNA sample purification kit, the MasterPure Complete DNA and RNA purification kit, the QIAamp DNA blood mini kit, and the UltraClean Microbial DNA isolation kit. The methods were compared using 6 F. tularensis strains representing the 2 subspecies which cause the majority of reported cases of tularemia in humans. Cell viability testing of the DNA extracts showed that all 6 extraction methods efficiently inactivated F. tularensis at concentrations of ≤10⁶ CFU/mL. Real-time PCR analysis using a multitarget 5′ nuclease assay for F. tularensis revealed that the PCR sensitivity was equivalent using DNA extracted by the 2 automated methods and the manual MasterPure and QIAamp methods. These 4 methods resulted in significantly better levels of detection from bacterial suspensions and performed equivalently for spiked swab samples than the remaining 2. This study identifies optimal DNA extraction methods for processing swab specimens for the subsequent detection of F. tularensis DNA using real-time PCR assays. Furthermore, the results provide diagnostic laboratories with the option to select from 2 automated DNA extraction methods as suitable alternatives to manual methods for the isolation of DNA from F. tularensis.     

Molecular beacons: colorful analysis of nucleic acids

The completion of the Humane Genome Project has resulted in an exponential rise in the demand for molecular diagnostic assays. To meet this demand, several innovative technologies have become available for performing homogeneous genetic analyses. For this type of assay, special detector probes are necessary. In 1996, Tyagi and Kramer described fluorogenic hairpin-shaped detector probes, called 'molecular beacons', which are extraordinarily specific. Since they characterize alleles by the generation of fluorescent signals, they are perfectly suited for homogeneous genetic analysis. Molecular beacons assays are simple, fast, inexpensive, sensitive, utilize a high-throughput format, enable the testing of many samples simultaneously and allow the detection of a series of different agents in the same assay tube. This review is designed to give the reader a greater understanding of the exciting applications of molecular beacons in DNA, RNA and protein studies.     

High-throughput HBV DNA and HCV RNA detection system using a nucleic acid purification robot and real-time detection PCR: its application to analysis of posttransfusion hepatitis

BACKGROUND: A high-throughput detection system was developed for HBV DNA and HCV RNA. METHODS: A combination of real-time detection PCR using an automated system (PRISM 7700, PE Biosystems, Foster City, CA) and automatic viral nucleic acid extraction (BioRobot 9604, Qiagen, Hilden, Germany) was used as the high-throughput detection system. An internal control for HBV DNA detection was also developed. RESULTS: Testing of 96 samples for HBV and HCV was completed within 5 hours. The sensitivity of this system almost equals that of the manual method using nested PCR. The addition of an internal control for HBV detection did not affect the sensitivity of the method and confirmed the accuracy of results. It was possible to quantify HBV in HBV+ samples that contain more than 500 genome equivalents per mL. We started using this system from June 1999 for testing stored donor and patient samples to analyze cases of posttransfusion hepatitis and identified three HBV+ donations that were implicated in posttransfusion hepatitis B. CONCLUSION: The high-throughput detection system is a useful tool for HBV DNA and HCV RNA detection because it enables rapid and reliable testing of a large number of samples.     

自动提取大样本全血基因组DNA方法的建立

目的建立从大样本全血中自动提取基因组DNA的方法,并评价提取DNA的质量和产量。方法基因组DNA使用MiniPrep75-Ⅱ自动工作站提取;DNA样本的纯度和含量用紫外分光光度法测定;DNA的完整性用琼脂糖电泳法测定。结果从100μl全血中平均可提取到(7.33±2.58)μg基因组DNA;DNA样本的纯度平均为1.647±0.135(A260/A280);琼脂糖电泳法测得DNA的分子量约为21kb。结论本方法可以快速从大样本全血中自动提取较高质量的基因组DNA,所得DNA适用于下游的分子生物学实验。     

Basic principles of real-time quantitative PCR

Real-time quantitative PCR allows the sensitive, specific and reproducible quantitation of nucleic acids. Since its introduction, real-time quantitative PCR has revolutionized the field of molecular diagnostics and the technique is being used in a rapidly expanding number of applications. This exciting technology has enabled the shift of molecular diagnostics toward a high-throughput, automated technology with lower turnaround times. This article reviews the basic principles of real-time PCR and describes the various chemistries available: the double-stranded DNA-intercalating agent SYBR Green 1, hydrolysis probes, dual hybridization probes, molecular beacons and scorpion probes. Quantitation methods are discussed in addition to the competing instruments available on the market. Examples of applications of this important and versatile technique are provided throughout the review.     

Robotic automation of coagulation analysis

Laboratory automation systems (LAS) have been installed in over 22 sites across North America providing automation of many preanalytical and analytical tasks in clinical laboratories. Only a few laboratories have automated the analysis of citrated whole blood for the diagnosis of hemostasis disorders. The analysis of coagulation factors in citrated blood requires a large amount of labor in order to provide rapid turnaround; thus automation of this analytical process is attractive. Therefore, we have created an automated coagulation workstation using a systematic approach to automation design and engineering. First, we used discrete event simulation to calculate potential throughput and to identify possible bottlenecks for the proposed coagulation workcell. We then created a three-dimensional animated computer model of the workstation to simplify workstation design. Finally, we constructed a prototype workcell using a mobile robot, an articulated robotic arm, and a coagulation analytical system.     

Capillary and microchip electrophoresis for rapid detection of known mutations by combining allele-specific DNA amplification with heteroduplex analysis

BACKGROUND: Detection of mutations by gel electrophoresis and allele-specific amplification by PCR (AS-PCR) is not easily scaled to accommodate a large number of samples. Alternative electrophoretic formats, such as capillary electrophoresis (CE) and microchip electrophoresis, may provide powerful platforms for simple, fast, automated, and high-throughput mutation detection after allele-specific amplification. METHODS: DNA samples heterozygous for four mutations (185delAG, 5382insC, 3867G-->T, and 6174delT) in BRCA1 and BRCA2, and homozygous for one mutation (5382insC) in BRCA1 and two mutations (16delAA and 822delG) in PTEN were chosen as the model system to evaluate the capillary and microchip electrophoresis methods. To detect each mutation, three primers, of which one was labeled with the fluorescent dye 6-carboxyfluorescein and one was the allele-specific primer (mutation-specific primer), were used to amplify the DNA fragments in the range of 130-320 bp. AS-PCR was combined with heteroduplex (HD) analysis, where the DNA fragments obtained by AS-PCR were analyzed with the conditions developed for CE-based HD analysis (using a fluorocarbon-coated capillary and hydroxyethylcellulose). The CE conditions were transferred into the microchip electrophoresis format. RESULTS: Three genotypes, homozygous wild type, homozygous mutant, and heterozygous mutant, could be identified by CE-based AS-PCR-HD analysis after 10-25 min of analysis time. Using the conditions optimized with CE, we translated the AS-PCR-HD analysis mutation detection method to the microchip electrophoresis format. The detection of three heterozygous mutations (insertion, deletion, and substitution) in BRCA1 could be accomplished in 180 s or less. CONCLUSIONS: It is possible to develop a CE-based method that exploits both AS-PCR and HD analysis for detecting specific mutations. Fast separation and the capacity for automated operation create the potential for developing a powerful electrophoresis-based mutation detection system. Fabrication of multichannel microchip platforms may enable mutation detection with high throughput.     

Quantitation of residual WBCs in filtered blood components by high-throughput, real-time kinetic PCR

BACKGROUND: The effort to eliminate transfusion complications associated with WBCs has led to the widespread use of filters able to reduce WBC concentrations to 相似文献   

全自动工作站与两种人工抽提全血基因组 DNA 方法的比较

目的比较全自动工作站与两种人工法抽提全血基因组DNA的质和量,总结三种方法的特点及提供一些建议。方法三种方法均选取同样36份样本抽提基因组DNA,并比较其浓度和纯度,并计算三种方法所需的费用。结果三种方法提取的基因组DNA均能达到相关分子生物学实验的要求,但在操作程序、价格和全血需要量等方面都存在较大差异。结论全自动工作站用全血量少,价格比较便宜,适合大批量全血样本抽提基因组DNA。Gentra法和胍盐酸法均适用于新鲜全血和冻融过的全血,胍盐酸法还适用于有血凝块的全血。     

Evaluation of a new pooling strategy based on leukocyte count for rapid quantification of allele frequencies

BACKGROUND: Allele frequencies of single-nucleotide polymorphisms (SNPs) can be quantified from DNA pools. The conventional preparation of DNA pools requires DNA isolation and quantification for each blood sample. We hypothesized that pooling of whole blood samples according to their leukocyte count, which determines DNA content, would be as reliable as the conventional pooling method but much less tedious to perform. METHODS: We collected 100 whole blood samples and measured the leukocyte count. Samples were frozen until further use. After thawing, pools were generated by combining aliquots containing an equal number of leukocytes. In parallel, DNA was extracted from another aliquot, DNA concentration was measured, and DNA concentration-based pools were assembled. All original samples were genotyped directly using 4 different SNP assays to obtain the exact allele frequencies in the pool. In addition, samples of known genotypes were mixed according to the DNA concentration or the leukocyte count to generate artificial samples of known allele frequencies. We analyzed pools and mixes in triplicate by pyrosequencing and calculated allelic frequencies. RESULTS: Leukocyte and DNA pooling provided equally accurate and precise SNP frequencies comparable to published data. CONCLUSION: DNA and leukocyte pooling are both suitable strategies to determine allele frequencies in frozen samples. The leukocyte pooling approach is much less tedious, quicker, and less expensive. It should be always considered if leukocyte counts are available.     

Bel亚型与α1,3半乳糖基转移酶基因G952A多态性关系的研究

目的 研究红细胞ABO血型系统中B放散型的ABO基因分子遗传基础.方法 通过标准血型血清学试验鉴定了3例Bel亚型及15例对照B型样本,采用ABO基因分型PCR序列特异性引物、ABO基因第6及第7外显子PCR产物直接测序及克隆测序等方法进行ABO基因及亚型的定型.结果 在1例血型血清学检测为Bel亚型标本中,发现一个新的B等位基因.该等位基因与B101标准等位基因相比,差异仅在于ABO基因的第7外显子上nt952位G>A突变,导致多肽链Val318Met,定为B放散型(Bel)新基因,GenBank注册号为EF117687.而其余2例Bel型样本及15例对照B型样本含正常标准B基因.结论 首次在ABO基因编码区核苷酸930位后的错义突变中发现并报道了新B等位基因,表明1,3半乳糖基转移酶基因G952A多态性可能是Bel分子遗传机制之一.     

A Novel Variant B Allele of the ABO Blood Group Gene Associated with Lack of B Antigen Expression

SUMMARY: BACKGROUND: The gene locus for the ABO blood group system encodes a glycosyltransferase. Alterations in the DNA sequence are associated with the blood groups and the expression levels of antigens on red blood cells. A number of ABO alleles have been described as the molecular basis of weak A or B antigens. PATIENTS AND METHODS: Here, we describe a novel variant B allele in a blood donor with discrepant results in routine forward (group A) and reverse (very weak anti-B isoagglutinins) ABO blood grouping. RESULTS: Determination of the ABO genotype using polymerase chain reaction-sequence-specific primers (PCR-SSP) indicated blood group A(2)B. Sequencing of the ABO gene exons 6 and 7 showed for 1 allele a G insertion into the GGGGGG sequence at position 811-816 of exon 7. The 816insG mutation (designated ABO*Bw20) led to a frame shift of the coding sequence and subsequent alteration of the protein sequence. The location of the mutation on a B allele was proven by PCR-SSP. Screening for the novel mutation in 211 blood donors with regular ABO phenotypes indicated that *Bw20 is a rare variant. CONCLUSIONS: The low levels of anti-B isoagglutinins associated with this novel variant indicate that residual undetectable amounts of B antigen may be present on red blood cells. The serological and molecular analysis of members of the blood donor's family further proved the phenotype-genotype correlation of the *Bw20 allele with antigen O and individually variable levels of anti-B isoagglutinins. The characterization of novel alleles associated with ABO subgroups may ensure the correct determination of blood groups in which serological methods are combined with molecular genetic approaches.     

Optimized linkage and quenching strategies for quantum dot molecular beacons

Quantum dot (QD) molecular beacons were explored for sequence-specific DNA detection. The effectiveness of multiple linkage strategies and fluorescence quenchers were compared in hybridization-based assays. To compare linkage strategies, covalent amide linkage and streptavidin-biotin binding were used to link semiconductor QDs to molecular beacon DNA. Amide-linked beacons showed a 57% greater fluorescence increase than streptavidin-linked beacons when hybridized to 200 pmol of target DNA. The specificity of the molecular beacons, however, was similar for both linkage methods. Hybridization of both QD molecular beacons with non-complementary target DNA resulted in approximately 50% lower fluorescence intensity than hybridization with complementary DNA. The effectiveness of different quencher moieties was also evaluated. Iowa Black and 1.4 nm Nanogold-quenched molecular beacons exhibited approximately 2-fold greater fluorescence increases than dabcyl-quenched beacons when hybridized to complementary target. Specificity for target DNA was also confirmed through hybridization assays with non-complementary DNA. To provide insight into differences between the QD molecular beacons and the linkage strategies used, the hydrodynamic radius of each was measured. These measurements indicated that the larger radius of the streptavidin QDs (13.5 nm) than the carboxyl QDs (7 nm) could have a negative effect on FRET-based quenching for QD molecular beacons. These data outline the importance of choosing proper linkage methods and quencher moieties for creating high-quality QD molecular beacons.