Towards a "Sample-In, Answer-Out" Point-of-Care Platform for Nucleic Acid Extraction and Amplification: Using an HPV E6/E7 mRNA Model System

Abstract

The paper presents the development of a "proof-of-principle" hands-free and self-contained diagnostic platform for detection of human papillomavirus (HPV) E6/E7 mRNA in clinical specimens. The automated platform performs chip-based sample preconcentration, nucleic acid extraction, amplification, and real-time fluorescent detection with minimal user interfacing. It consists of two modular prototypes, one for sample preparation and one for amplification and detection; however, a common interface is available to facilitate later integration into one single module. Nucleic acid extracts (n = 28) from cervical cytology specimens extracted on the sample preparation chip were tested using the PreTect HPV-Proofer and achieved an overall detection rate for HPV across all dilutions of 50%-85.7%. A subset of 6 clinical samples extracted on the sample preparation chip module was chosen for complete validation on the NASBA chip module. For 4 of the samples, a 100% amplification for HPV 16 or 33 was obtained at the 1 : 10 dilution for microfluidic channels that filled correctly. The modules of a "sample-in, answer-out" diagnostic platform have been demonstrated from clinical sample input through sample preparation, amplification and final detection.

Figure 1

Schematic approach to the development of an integrated LOC device for the detection of HPV nucleic acid (involving a nucleic acid extraction chip and NASBA amplification chip).

Figure 2

Automated LOC system for sample preconcentration, nucleic acid extraction, amplification, and real-time fluorescent detection. (a) Sample preparation chip: sample inlet [1], cell filter [2], a silica phase extraction filter [3], nucleic acid extraction reagent storage chamber [4A], storage chamber for DMSO and sorbitol [4B] turning valves [5], waste outlet [6], sample outlet [7], and a pressure sensor [8] (b) Sample preparation instrument (c) NASBA chip: sample inlet [1], supply channel [2], metering channels [3], valve 1 [4A], valve 2 [4B], valve 3 [4C], chamber 1 [5A], chamber 2 [5B], waste chamber [6], and instrument connection interface [7] (d) NASBA instrument.

Figure 3

(a) The amplification plot of the clinical sample 508 (HPV 33, 1 : 10 dilution). All the 7 reaction chambers filled with sample are classified as positive using our inhouse data analysis code also used in the conventional PreTect Analyser instrument. Reaction chamber number 8 was not filled with sample; hence, no increase of fluorescence was observed. The offset of the curve is also lower than the filled chambers, as the autofluorescence of the reaction mixture was not present. (b) Fluorescent micrograph of reaction chambers (8–1) after amplification of clinical sample 508 (HPV 33, 1 : 10 dilution). The air bubble located in reaction chamber number 4 contributes to the lower fluorescent signal detected by the NASBA instrument. No sample has entered reaction chamber 8.

Figure 4

Two colour detection of fluorescence from NASBA amplified products using the Multiplex Detector Version 2 (a) Detection of HPV 16-FAM and U1A-ROX, Channels 1, 5, and 6 were empty. (b) Detection of HPV 18-ROX and HPV 31-FAM, Channel 2 empty. (c) Detection of HPV 33-FAM and HPV 45-ROX, Channels 4 and 8 empty.

Figure 5

U1A and HPV16 amplification plots from NASBA chips containing freeze-dried reagents. Freeze-dried primers and probes (U1A/HPV16) were deposited in reaction chamber 1 (250 nL) and enzymes were deposited in reaction chamber 2 (125 nL). Incubation time in reaction chamber 1 was 5 minutes. The chips were stored at room temperature for 8 and 9 days. (a) Positive control U1A/HPV 16. Channel 8 was empty. (b) CaSki (1 : 10 dilution, equivalent to 370 cell/reaction chamber on chip). Channel 6 was empty. (c) HPV 16 positive clinical specimen (Pretoria). (d) No template control. Channel 7 was empty.

Figure 6

Amplification plot of HPV16 and U1A performed on the NASBA chip with freeze-dried enzymes in reaction chamber 2 stored at room temperature for 32 days. Channel 1 was empty. An instrumental error resulted in an outlier measurement for HPV16 in channel 5 after 88 minutes.