From Single Cell Gene-based Diagnostics to Diagnostic Genomics: Current Applications and Future Perspectives

Clinical Laboratory Science, Fall 2005 by Zhao, Richard

One advantage of using NASBA is that it can use any types of specimen, e.g., serum, plasma, cerebrospinal fluid, tissue, seminal plasma, or genital secretions. There is no specific requirement for anticoagulant. A drawback of this test is that it requires a separate RNA extraction step, making it more labor intensive. Consequently, a potential problem with NASBA is the significant risk of carry-over contamination between samples since much pipetting and tube opening must be performed for the isolation of viral RNA from plasma or serum specimens. The commercial name of NASBA amplification products is NucliSens(TM) through bioMerieux, Inc. Commercial tests currently are available include NucliSens CMVpp67, RSVA B, Enterovirus, and HIV-I.

PROBE-BASED AMPLIFICATION

Ligase chain reaction (LCR)

In contrast to target-based amplification technology, probebased technique is designed to amplify the probe homologous to the target. LCR is a method designed to amplify the probes that are homologous to a specific target of interest. The rationale behind this method is that ligation is most efficient when two discrete probes hybridize to the target in a

head-to-tail fashion. Once the first pair of probes is ligated, it can in turn serve as a template for annealing and ligation of a second pair of probes complementary to its sequence. Recycled ligations between these two sets of ligated probes plus the original targets will result in a logarithmic accumulation of ligation products. Diagnostic tests using LCR are developed under the trademark name of LCx at Abbott Laboratories. A HIV-I LCx commercial test is available outside the United States.

Strand displacement amplification (SDA)

The key principle of SDA resembles DNA excision DNA repair.9 In the presence of a single strand nick on a double stranded DNA molecule, the nicked strand will be prejudicially displaced by a newly synthesized DNA strand. If it were possible to create a nick repeatedly at the same location, displaced DNA strands of the same size could then be produced continuously. To accomplish this goal, an endonuclease restriction enzyme Hindi or BsoBl is used, which only nicks DNA on one strand of its recognition site (5'-GTTGAC-3') when the opposite strand is hemiphosphorothiolated (DNA contains dATPαS instead of dATP) during DNA synthesis. The primers used in this method contain this particular recognition site and are designed in such a way that the size of the displaced strand will be the same as the target. Therefore, the displaced DNA strands can also be used as templates for subsequent reactions. Repetition of this nicking and displacement cycle will result in a geometric accumulation of the synthetic product with approximately a 10^sup 8^-fold amplification within two hours. Although this method has the potential to be used in diagnosis of many diseases such as HIV-I and Mycobacterium spp, no commercial tests have yet been marketed.

Qβ-replicase amplified probe assay

Qβ-replicase amplified probe assay is another probe-based amplification method. This method takes advantage of a unique feature of the bacteriophage Qβ-replicase, which replicates only those RNA substrates with a specific secondary structure. Therefore, by incorporating a target-specific probe into a Qβ-replicase substrate, not only does this probe-containing substrate hybridize to the gene target, but the Qβ-replicase specifically amplifies only the hybridized RNA substrates. Unhybridized probes will be removed upon RNaseIII treatment. Using this method, one copy of the target could be amplified into 10^sup 9^ copies with 30 minute incubation. The use of Qβ-replicase amplified probe assay has been described previously by Gene-Track Inc. for the detection of HIV, C. trachomatis, M. avium, and M. tuberculosis. However, no commercial kits are yet available using this technology.