Analytical Performance Characteristics of the Cepheid GeneXpert Ebola Assay for the Detection of Ebola Virus.

BACKGROUND: The recently developed Xpert® Ebola Assay is a novel nucleic acid amplification test for simplified detection of Ebola virus (EBOV) in whole blood and buccal swab samples. The assay targets sequences in two EBOV genes, lowering the risk for new variants to escape detection in the test. The objective of this report is to present analytical characteristics of the Xpert® Ebola Assay on whole blood samples. METHODS AND FINDINGS: This study evaluated the assay's analytical sensitivity, analytical specificity, inclusivity and exclusivity performance in whole blood specimens. EBOV RNA, inactivated EBOV, and infectious EBOV were used as targets. The dynamic range of the assay, the inactivation of virus, and specimen stability were also evaluated. The lower limit of detection (LoD) for the assay using inactivated virus was estimated to be 73 copies/mL (95% CI: 51-97 copies/mL). The LoD for infectious virus was estimated to be 1 plaque-forming unit/mL, and for RNA to be 232 copies/mL (95% CI 163-302 copies/mL). The assay correctly identified five different Ebola viruses, Yambuku-Mayinga, Makona-C07, Yambuku-Ecran, Gabon-Ilembe, and Kikwit-956210, and correctly excluded all non-EBOV isolates tested. The conditions used by Xpert® Ebola for inactivation of infectious virus reduced EBOV titer by ≥6 logs. CONCLUSION: In summary, we found the Xpert® Ebola Assay to have high analytical sensitivity and specificity for the detection of EBOV in whole blood. It offers ease of use, fast turnaround time, and remote monitoring. The test has an efficient viral inactivation protocol, fulfills inclusivity and exclusivity criteria, and has specimen stability characteristics consistent with the need for decentralized testing. The simplicity of the assay should enable testing in a wide variety of laboratory settings, including remote laboratories that are not capable of performing highly complex nucleic acid amplification tests, and during outbreaks where time to detection is critical.

Development of an integrated assay for detection of BCR-ABL RNA.

BACKGROUND: Current practice guidelines for managing patients with chronic myelogenous leukemia (CML) call for monitoring BCR-ABL transcript concentrations with a quantitative reverse transcription-PCR (qRT-PCR) assay. Because the available laboratory-developed assays lack consensus on the appropriate design, reporting of results, and reference intervals, we developed and evaluated an integrated BCR-ABL assay that yields standardized results for any laboratory and can be performed by technicians with no specialized training. METHODS: We used the Cepheid Xpert BCR-ABL Monitor assay to measure both BCR-ABL and ABL (endogenous control) transcripts in blood samples from CML patients and healthy individuals. The assay involves 8 manual pipetting steps, fully automated nucleic acid purification, a nested qRT-PCR step, and data analysis. RESULTS: The BCR-ABL assay requires approximately 2 h 20 min and covers a 5-log concentration range with a lower detection limit for the BCR-ABL:ABL ratio of approximately 0.005%. Assay results were negative for 100% of the 56 known CML-negative samples (12 patients with other hematologic disorders and 44 healthy blood donors). Testing of CML-positive patients undergoing disease monitoring showed 85% agreement with negative results (17 of 20) and 100% agreement with positive results (26 of 26). An imprecision/portability study revealed no differences in performance between sites, days, instruments, and operators. CONCLUSIONS: The Xpert BCR-ABL Monitor assay provides a robust and reproducible alternative to laboratory-developed assays. Its ease of use may allow more laboratories to offer BCR-ABL testing for patients, and the short assay time enables same-day results for treating physicians.

Evaluation of the Cepheid GeneXpert BCR-ABL assay.

Patients with chronic myeloid leukemia harbor the chromosomal translocation t(9;22), which corresponds to fusion of the BCR and ABL genes at the DNA level. The translated fusion product is an oncogenic protein with increased ABL tyrosine kinase activity causing cell transformation. To date, reverse transcriptase-polymerase chain reaction is considered the most sensitive method available for detecting low copy numbers of the BCR-ABL gene fusion. Recently, Cepheid introduced its GeneXpert-based assay for the identification of the BCR-ABL gene fusion in cells from blood samples. This system comprises a walk-away self-contained instrument that combines cartridge-based microfluidic sample preparation with reverse transcriptase-polymerase chain reaction-based fluorescent signal detection and BCR-ABL and ABL Ct (threshold cycle) determination. The difference between the BCR-ABL Ct and ABL Ct (DeltaCt) is expected to represent the ratio of the two populations of mRNAs and ultimately the percentage of neoplastic cells present. We tested whether this BCR-ABL fusion detection system could be used as a clinical diagnostic tool for monitoring patients with minimal residual disease of chronic myelogenous leukemia. We report similar performance characteristics, including limit of detection, specificity, sensitivity, and precision, of this automated BCR-ABL fusion detection system to those of a manual TaqMan reverse transcriptase-polymerase chain reaction-based test.

Photocrosslinking oligonucleotide hybridization assay for concurrent gene dosage and CpG methylation analysis.

The phenotypic effects of aberrant gene expression are indistinguishable, regardless of whether the underlying mutation is one of gene copy number (deletion or duplication) or modification of differentially methylated CpG sites occurring in critical regulatory sequences in gene promoters. The XLnt photocrosslinking oligonucleotide technology provides for the hybridization-dependent covalent attachment of probe to target, allowing survival of the probe/target complex under otherwise denaturing conditions. Posthybridization wash stringency can be substantially higher than under standard techniques, leading to a marked increase in signal-to-noise ratios. In addition, the reduction in nonspecific background provides linearity to XLnt-based oligonucleotide assays comparable to that otherwise achieved only with very long probes. The technology is thus ideally suited for combining the high-throughput capacity of oligonucleotide hybridization platforms with accurate measurement of relative gene dosage. By integrating the XLnt system with an assay design separating probe/target immobilization and signal elaboration functions, relative gene dosage assessment can be applied to the quantitation of fractional resistance to methylation-sensitive restriction enzyme (MSRE) digestion. The method described below provides for the development of photocrosslinking oligonucleotide assays for relative gene dosage and fractional locus CpG methylation in a microtiter plate-based format.

Detection of the factor V Leiden mutation by a modified photo-cross-linking oligonucleotide hybridization assay.

BACKGROUND: Our previously developed assay for detection of the factor V Leiden mutation (G1691A) based on a nucleic acid photo-cross-linking technology used two allele-specific capture probes and six fluorescein-modified signal-generating reporter probes. We wished to improve the sensitivity and performance of the method. METHODS: We developed new reporter probes with approximately 10-fold more fluorescein molecules than the original probes. The single, cross-linker-modified capture probe was replaced by a three-probe system, separating the probe-target cross-linking function and the allelic differentiation function. The capture probe cross-linked to either or both of two flanking probes through stem structures at the capture-probe/flanking-probe junctions. The flanking probes cross-linked to target DNA through two cross-linking sites each. Genomic DNA was extracted from 0.2 mL of whole blood and restriction-enzyme digested to create a defined 677 bp target sequence. Preliminary genotype ranges were determined for the assay by testing of pre-typed samples. We then tested 1054 clinical samples, using an automated sample processor. RESULTS: The new assay had a 10-fold increase in signal-to-background ratio. Genotype results for 1039 of 1054 clinical samples (98.6%) agreed with those of a PCR-based method. Of the 15 remaining samples, 10 produced an indeterminate result outside the defined genotype ranges, 2 yielded insufficient signal to be genotyped, and 3 gave a discordant result. All 15 samples were genotyped correctly after re-extraction of genomic DNA and retesting. CONCLUSION: The modified photo-cross-linking assay for factor V Leiden detection is a sensitive non-PCR-based assay with potential for use in high-throughput clinical laboratories.

High-throughput detection of submicroscopic deletions and methylation status at 15q11-q13 by a photo-cross-linking oligonucleotide hybridization assay.

BACKGROUND: Current technologies for assessing genetic deletions and duplications of greater than one kilobase are labor-intensive or rely on PCR-based methods, and none offers the ability to simultaneously detect dosage abnormalities, assess 5'-to-3' cytosine-guanosine (CpG) methylation, and interrogate single-nucleotide polymorphisms (SNPs). We describe a high-throughput platform for direct gene-dosage determination capable of concurrent assessment of other forms of gene modification. METHODS: We used a light-activated interstrand nucleic acid cross-linking system (XLnt technology) to determine gene dosage at the 15q11-q13 deletion/duplication locus. We incorporated restriction enzyme digestion of genomic DNA into the method to assess CpG methylation in parallel with gene dosage. For method validation we used DNA from 31 cell lines with previously characterized 15q11-q13 gene dosage and parental origin status. Diagnostic cutoffs were set at 0.5 +/- 0.15, 1 +/- 0.15-0.25, and 2 +/- 0.3. RESULTS: Dosage-only experiments showed discrimination of deletions (n = 21) from healthy controls (NCs; n = 27) in all samples. Five of 49 samples gave results outside of specification. Concurrent evaluation of dosage and CpG methylation yielded dosage results within specification for 18 of 19 deletion and 8 of 12 NC samples. Paternal deletion and NC methylation pattern results were within specification in 17 of 19 and 9 of 12 runs, respectively. No overlap was demonstrated between value sets for the two groups. CONCLUSIONS: The XLnt technology provides a rapid, high-throughput platform for the accurate determination of gene dosage. The flexibility of this technology allows parallel interrogation of gene dosage, CpG methylation, and SNPs.