Alternative molecular tests for virological diagnosis

Abstract

Several nucleic acid amplification techniques (NAATs), particularly PCR and real-time PCR, are currently used in the routine clinical laboratories. Such approaches have allowed rapid diagnosis with a high degree of sensitivity and specificity. However, conventional PCR methods have several intrinsic disadvantages such as the requirement for temperature cycling apparatus, and sophisticated and costly analytical equipments. Therefore, amplification at a constant temperature is an attractive alternative method to avoid these requirements. A new generation of isothermal amplification techniques are gaining a wide popularity as diagnostic tools due to their simple operation, rapid reaction and easy detection. The main isothermal methods reviewed here include loop-mediated isothermal amplification, nucleic acid sequence-based amplification, and helicase-dependent amplification. In this review, design criteria, potential of amplification, and application of these alternative molecular tests will be discussed and compared to conventional NAATs.

Fig. 1

Schematic representation of primer design for LAMP assay. The figure shows the position of six primers (FIP, BIP, F3, B3, Loop F, Loop B) spanning the target gene

Fig. 2

Schematic representation of NASBA process using molecular beacons probes as a detection system. The figure shows the two phases of the NASBA amplification process characterized by a linear and exponential kinetics

Fig. 3

Amplification scheme of HDA method. (Step 1) DNA helicase unwinds double-stranded DNA. (Step 2) SSB proteins stabilize the displaced DNA strands. (Step 3) Specific primers hybridize to the ssDNA template and are extended by DNA polymerase. (Step 4) A double-stranded copy of the DNA target is produced