University Honors

Huanglongbing (HLB), also known as citrus greening disease, is transmitted by the Asian citrus psyllid and infects citrus trees, leading to leaf mottling, poorly colored and bitter-tasting fruit, and ultimately, tree death. The causative agent for HLB is Candidatus Liberibacter, a gram-negative, alpha-proteobacterium. While no treatment for HLB currently exists, the quantitative Polymerase Chain Reaction (qPCR) serves as a gold standard for detecting target genes. The qPCR system identifies the presence of our target gene through simultaneous thermocycling and detection. This study aimed to answer the question: Can we reduce the cycle threshold (Ct) value in qPCR runs without increasing the initial concentration of nucleic acid? We proposed two methods to lower Ct values. The first method involved using ribonucleic acid (RNA) instead of deoxyribonucleic acid (DNA), with reverse transcription converting RNA to complementary DNA (cDNA). The second method utilized protein additives such as T4 Gene 32 Protein, RecA, and ET-SSB. Dialysis of the protein sample was performed to test the difference between the stock solution and the dialyzed counterpart. The substitution of DNA with RNA resulted in an enhancement in signal detection, yielding a difference of approximately 1.5 in the cycle threshold at high template concentrations and about 4.9 at low template concentrations. However, the effects of proteins did not meet our expectations due to various factors that could potentially influence the interpretation of the collected data. This unexpected outcome opens a new avenue for experimentation with different factors to validate these results. The goal of this project is to improve the detection of signal at the minimal amount of genetic material, thereby enabling the detection of the presence of infected plant hosts before they spread to healthy host plants.