Point-of-care nucleic acid testing for infectious diseases

Abstract

Nucleic acid testing for infectious diseases at the point of care is beginning to enter clinical practice in developed and developing countries; especially for applications requiring fast turnaround times, and in settings where a centralized laboratory approach faces limitations. Current systems for clinical diagnostic applications are mainly PCR-based, can only be used in hospitals, and are still relatively complex and expensive. Integrating sample preparation with nucleic acid amplification and detection in a cost-effective, robust, and user-friendly format remains challenging. This review describes recent technical advances that might be able to address these limitations, with a focus on isothermal nucleic acid amplification methods. It briefly discusses selected applications related to the diagnosis and management of tuberculosis, HIV, and perinatal and nosocomial infections.

Figure 1

Selected sample preparation technologies for POC NAT. (a) Detailed schematic of Cepheid’s GeneXpert cartridge reproduced with permission from [10], most of which is devoted to sample preparation and reaction setup. A plunger from the instrument engages with the syringe barrel to draw sample and lysis/binding, wash, and elution buffers through the rotating valve body at the bottom of the cartridge into the cavity that holds beads for NA SPE. Eluted, purified nucleic acids are combined with lyophilized mastermix reagents and transferred into the PCR chamber at the side of the cartridge for amplification with real-time fluorescence-based detection. (b) Miniaturized ultrasonic horn incorporated into the GeneXpert system reproduced with permission from [16], which engages with the bottom of the cartridge to facilitate pathogen lysis. (c) Miniaturized bead blender developed by Claremont BioSolutions [17] to mechanically disrupt lysis-resistant pathogens. (d) “No wash” sample preparation, based on moving nucleic acids bound to magnetic beads from the lysis/binding buffer on the left through a layer of liquid wax into the elution buffer chamber on the right, reproduced with permission from [19].

Figure 2

Overview of isothermal nucleic acid amplification reactions. (a) Methods based on RNA transcription. In TMA (Gen-Probe) [29] and NASBA (BioMerieux) [30], an RNA target is converted to ds cDNA with a promoter region through reverse transcription, followed by RNase H degradation of the original strand and DNA polymerization initiated by a second primer. RNA polymerase (pol) amplification creates products that feed back into the original reaction. TMA and NASBA involve the same reaction scheme, but NASBA requires three enzymes (RT-DNA pol, RNase H, and RNA pol); TMA requires only two enzymes, because the RT-DNA pol has intrinsic RNase H activity. SMART (Cytocell) [31] utilizes a three-way junction with target and extension probes to initiate linear RNA polymerization based amplification (no exponential feedback). (b) Methods based on DNA replication with enzymatic duplex melting/primer annealing. In HDA (Biohelix) [32,33], a helicase enzymatically “melts” dsDNA. In RPA (TwistDx) [34,35], a recombinase–primer complex scans dsDNA for the target site and facilitates primer binding. In both HDA and RPA, single strand binding proteins stabilize the separated strands; the rest of the reaction sequence is analogous to PCR. (c) Methods based on strand displacement using polymerases only from a linear target, through use of sacrificial outer bumper primers. LAMP (Eiken) [36,37] involves six recognition sites on the target DNA; the 5′ overhangs of the inner primers recognize sequences in the amplicon, which leads to the generation of a dumbbell structure. CPA (Ustar) [38] involves four recognition sites on the target DNA, and the inner primers lead to cross-priming after the first round. SMART-AMP (Riken Institute) [39] includes five recognition sites on the target DNA as well as a fifth booster primer; single nucleotide polymorphism discrimination is facilitated through use of the MutS protein. (d) Methods based on strand-displacing polymerization from inherently circular targets in RCA (Molecular Staging) [40], or from padlock probes that are circularized through action of a ligase in RAM (Thorne Diagnostics) [41]. In both cases, branched amplification can be initiated through a second primer.

Figure 3

Isothermal nucleic acid amplification methods that are based on polymerase extension plus a single-strand cutting event. In SDA (Becton Dickinson) [42,43], NEMA (Ustar) [45] and ICA (RapleGene) [46], an intermediate target is generated using strand-displacing amplification via sacrificial outer bumper primers. NEAR (Ionian Technologies) [44] does not use bumper primers and involves shorter amplicons than the other methods. In SDA, phosphothioates are incorporated into the amplicon during polymerization so that a restriction endonuclease only cuts one strand. NEAR and NEMA both use nicking endonucleases that are inherently single strand cutting. In ICA, a single strand cut is facilitated through RNase H and DNA–RNA–DNA chimeric primers. After the single-strand cutting event, amplicons are generated through strand-displacing amplification, for short amplicons further facilitated by thermal denaturation. These amplicons re-prime, and lead to exponential feedback amplification. EXPAR [47] amplifies short trigger oligonucleotides, which can be generated via the so-called “fingerprinting” reaction [48] from adjacent nicking enzyme recognition sites in genomic DNA. This is followed by rapid exponential amplification, mediated by a template sequence that contains two copies of the trigger complement that are separated by the nicking enzyme recognition site complement. BAD-AMP [49] uses a molecular beacon for signal generation and as a template for single-strand nicking and re-priming.

Figure 4

(a) Antibody-dependent NALF, which uses antibody-conjugated colored particles and LF strips, and also requires an antigen-functionalized capture oligonucleotide and a target amplicon (blue) that contains an antigen. (b) Antibody-independent NALF involves direct hybridization of unlabeled target amplicon (blue) with oligonucleotide-functionalized colored particles and with oligonucleotides deposited on the LF strip.