Michelle L. Curtis

An invasive cleavage assay for direct quantitation of specific RNAs

RNA quantitation is becoming increasingly important in basic, pharmaceutical, and clinical research. For example, quantitation of viral RNAs can predict disease progression and therapeutic efficacy. Likewise, gene expression analysis of diseased versus normal, or untreated versus treated, tissue can identify relevant biological responses or assess the effects of pharmacological agents. As the focus of the Human Genome Project moves toward gene expression analysis, the field will require a flexible RNA analysis technology that can quantitatively monitor multiple forms of alternatively transcribed and/or processed RNAs (refs 3,4). We have applied the principles of invasive cleavage and engineered an improved 5′-nuclease to develop an isothermal, fluorescence resonance energy transfer (FRET)–based signal amplification method for detecting RNA in both total RNA and cell lysate samples. This detection format, termed the RNA invasive cleavage assay, obviates the need for target amplification or additional enzymatic signal enhancement. In this report, we describe the assay and present data demonstrating its capabilities for sensitive (<100 copies per reaction), specific (discrimination of 95% homologous sequences, 1 in ≥20,000), and quantitative (1.2-fold changes in RNA levels) detection of unamplified RNA in both single- and biplex-reaction formats.

Analytical performance of the Investigational Use Only Cervista™ HPV HR test as determined by a multi-center study

BACKGROUND Any HPV test designed to be utilized in cervical cancer screening programs should be highly validated both analytically and clinically. OBJECTIVES The Investigational Use Only (IUO) Cervista HPV HR test is designed to detect 14 high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68). The analytical performance of the Cervista HPV HR test was characterized in a multi-center study. RESULTS Analytical sensitivity for the 14 high-risk HPV types that the test is designed to detect ranged from 1,250 copies to 7,500 copies per reaction depending on HPV type. Accuracy compared to PCR with bi-directional sequencing was 91.4% [95% CI: 86.5 95.0%]. The reproducibility, when tested at three different testing centers, resulted in an overall inter-run reproducibility (between day/within site) agreement of 98.8% [1-sided 95% Confidence Lower Limit = 96.9%] and an overall inter-site reproducibility (between site) agreement of 98.7% [1-sided 95% Confidence Lower Limit = 97.9%]. The Cervista HPV HR test showed no cross-reactivity with DNA from seven non-oncogenic HPV types or 17 different infectious agents at up to 10(7) copies per reaction. CONCLUSIONS The analytical performance of the Cervista HPV HR test demonstrates sufficient analytical performance for use in cervical cancer screening. As with any clinical laboratory test, analytical characteristics must be evaluated in light of the clinical performance of this assay.

Analytical performance of Cervista HPV 16/18 genotyping test for cervical cytology samples.

BACKGROUND Human papillomavirus (HPV) types 16 and 18 are the 2 most frequent types associated with cervical cancer. Identifying their presence or absence in cervical samples may assist in triaging women for subsequent management. The Cervista HPV 16/18 genotyping test specifically detects the presence of HPV 16 and 18 in ThinPrep cervical specimens. OBJECTIVES The objective was to establish the analytical performance of the CERVISTA HPV 16/18 genotyping test. STUDY DESIGN These studies were performed in support of a regulatory submission to the US Food and Drug Administration. Here we report the analytical sensitivity (limit of detection), accuracy compared to consensus L1 gene PCR/bi-directional sequencing, precision, reproducibility, and cross-reactivity (specificity) of the genotyping test. RESULTS Analytical sensitivity for detection of HPV 16 and 18 ranged between 625 and 1250 copies/reaction for both types. When compared to PCR/sequencing for women with atypical squamous cells of undetermined significance cytology, the positive percent agreement was 94.1% (95% confidence interval [CI], 89.8-96.7) and the negative percent agreement was 85.7% (95% CI, 82.4-88.4). The test demonstrated high within-laboratory and inter-operator precision. Reproducibility within sites and between 3 testing sites resulted in 100% agreement with expected results (150 positive, 90 negative results). The genotyping test did not exhibit cross-reactivity to DNA from common low-risk HPV types and other microorganisms found in the human female reproductive tract. CONCLUSIONS These analytical performance data support the use of CERVISTA HPV 16/18 genotyping test for the detection and differentiation of HPV 16 and 18 in ThinPrep cervical cytology specimens.

Invader Assay for RNA Quantitation

The Invader assay is a homogeneous, isothermal, signal amplification system for the quantitative detection of nucleic acids. The assay can directly detect either DNA or RNA without target amplification or reverse transcription. It is based on the ability of Cleavase enzymes to recognize as a substrate and cleave a specific nucleic acid structure generated through the hybridization of two oligonucleotides to the target sequence. The combination of sequence-specific oligonucleotide hybridization and structure-specific enzymatic cleavage results in a highly specific assay well suited for discriminating closely related gene sequences. This includes detection of single nucleotide polymorphisms directly from genomic DNA as well as highly homologous mRNAs in closely related gene families. Because Cleavase substrate recognition is structure, and not sequence dependent, cleavage and detection can be applied to virtually any DNA or RNA sequence.

Erratum: An invasive cleavage assay for direct quantitation of specific RNAs

Peggy S. Eis, Marilyn C. Olson, Tsetska Takova, Michelle L. Curtis, Sarah M. Olson, Tatiana I. Vener, Hon S. Ip, Kevin L. Vedvik, Christian T. Bartholomay, Hatim T. Allawi, Wu-Po Ma, Jeff G. Hall, Michelle D. Morin, Tom H. Rushmore, Victor I. Lyamichev, and Robert W. Kwiatkowski. Nat. Biotechnol. 19, 673–676 (2001).