Implemented Design Algorithms of Unique Probes and Minimum Set Primers: Towards Early Differential Detections Applied on Multigene Targets of Symptom-Related Pathogens

The construction of a highly-efficient and rapid detection of symptom-related pathogens has been an important issue in the prevention and control of epidemics. Actual case studies on RNA virus infections, such as Severe Acute Respiratory Syndrome (SARS), have proven what danger misdiagnosis and widespread infection may lead into. Due to the highly mutational nature of RNA viruses, traditional detections by primers often fail as false negative; the difficulty is increased by the complex target genes of symptom-related pathogens.

The PDA-UniQ/MS algorithm uses the “4-mer” hash table by Tetra-Nucleotide Nucleation (TNN) to implement the nearest-neighbor melting temperature calculation and the structure filters. Then, it uses the modified compact genetic algorithm (MCGA) to design the unique probe (uni-probe) and multiple-use primers (mu-primers). The implemented result shows PDA-UniQ/MS greatly reduce the amount of mu-primers needed. In our trial of 12669 amplified target sequences, the reduction rate may reach up to 68% of that of conventional experiments. Further, the result of extended simulation proves that PDA-UniQ/MS will reduce more mu-primers according to the increasing number of target sequences

The unique probes and minimum set of mu-primers produced by this PDA-UniQ/MS algorithm is applicable to high-sensitivity electrochemical biosensor platforms either with or without amplification. The benefit of this is that: first, the unique probes, 50-monomer oligonucleotides, can be used as both array probe and detector probe, applying the catalyzed reporter deposition (CARD) electrochemical technique; second, the minimum set of primers, 12-monomer oligonucleotides, applying the polymerase chain reaction (PCR) electrochemical technique, increases the reusability of primers on multiple target genes, which reduces the cost on primer production. Using non-target-related sequences to extra design an 8-monomer oligonucleotide linker for mu-primers, we may minimize the melting temperature range of primers in the same set to simplify the process of the experiment and improve the efficiency of a reaction.

Actual detection of plant viruses which are 9 particular representative genes among 4 genuses is done by using the minimum set of mu-primers to implement touch-down PCR on 12-monomer primer fragments and steady state PCR on the total 20-monomer mu-primer with linker to amplify the samples. The experimental result proves the 11 mu-primers can successfully amplify the 9 target genes of plant viruses instead of traditional 18 primers. It shows PDA-UniQ/MS can greatly reduce the cost of PCR experiment and improve both the quality and efficiency of the presenting technology. In the future, a hybridization experiment integrated with the labeled nucleotides of pathogen is the validation of electrochemistry biosensor.

Concerning the design of symptom-related pathogens, we noted that, as with the case of SARS virus, the high mutation rate of RNA viruses must be considered, or the detection would likely fail due to sequence mutation. Based on the biological characteristic of highly-conserved regions, PDA-UniQ/MS algorithm successfully designed 73 mu-primers to amplify 50 target sequences. It also produced unique detection probes according to biological structure characteristics and probe specificity, so that rapid detection may be achieved.