Progress 08/01/17 to 03/31/19
Outputs
Target Audience:
Nothing Reported
Changes/Problems: Aim 2: Implementation of a TempO-STAT Liberibacter assay with <2 hr turn-around time. Success Criteria: Dual sample TempO-STAT assay with <2 hr TAT and sensitivity comparable to, or better than, the reference q-PCR assay. Status: Completed, but not successful. The TempO-STAT protocol was established and the "taqman" probe design proposed in the application was tested and it was confirmed that it provided a suitable design using a set of lymphocyte genes selected as radiation exposure biomarkers and lysate sample from cultured cells. Fig 1 depicts exemplar data from the TempO-STAT assay. The Psyllid TempO-STAT assay was subsequently established. Psyllid genes were labeled with Hex, Candidatus Liberibacter (CLas) genes with FAM. The protocol was not successfully shortened, in part because we only had access to a standard BioRad qPCR instrument, but also in part due to difficulty obtaining sufficient reference RNA (see below) to carry out multiple experiments testing the Tempo-STAT step conditions to shorten each, and because one of the commercial reagents contained enzyme from a third party which contained an interfering substance, leading to using up reference RNA, and thus, preventing optimization of the TempO-STAT steps suing CLas RNA. Aim 3: Demonstrate the use of psyllid and leaf lysates for the TempO-STAT Liberibacter assay. Success Criteria: TempO-STAT and TempO-Seq data from lysates correlate to matched RNA R2>0.8, and provides equal or better sensitivity.** Status: Not successful. The TempO-STAT psyllid assay gave unusual qPCR traces, not consistent with the Aim 2 validation data depicted in Fig 1. Additional reference samples were shipped, but the same unusual tracings were obtained. The psyllid STAT traces were the same for reference samples which, when tested by qPCR, were positive (Fig 2a, HEX signal tracing) or negative (Fig 2b, Hex tracing) for CLas. That was expected. However, the CLas gene measurements (FAM) were not only early but low, but there was no difference between reference samples that was CLas positive (Fig 2am FAM signal) or CLas negative (Fig 2b, FAM signal). Thus, the TempO-STAT data did not agree with the benchmark qPCR data. In addition, subsequently to the completion of this program it was discovered that one of the commercial reagents contained enzyme from a third party which contained an interfering substance. It was planned to retest samples, but those plans were interrupted by COVID-19 and inability to work in the laboratory, as the research was deemed to be non-essential. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Executive Summary: Purpose - The major goals of this program are to establish anddemonstrate the feasibility of a multiplexed qPCR-like assay (TempO-STAT) and aTempO-Seq targeted sequencing assay to identify CLas infection in psyllids and citrus leaves. These tests will not require RNA to be extracted from the psyllids or leaves, nor require reverse transcription of the RNA, and will measure both CLas genes and response genes in psyllids and citrus leaves.The concept for wanting to establish anddemonstrate the feasibility of these tests is based on the following:the multiplexed qPCR-like assay will not require the extraction of RNA it can be carried out in the field by non-technical persons, infection-positivesamples from this test can then be sent to a central lab where itcan be verified, and additional information obtained regarding the severity and stage of infection by testing in the TempO-Seq assay. Should we be successful in demonstrating feasibility in this Phase Iwe will submit a Phase II application to produce abeta-test multiplexed qPCR-likeassay and platform and a beta-test TempO-Seq assay andperform initialfield testing. Description of Research Carried out - The design of probes for the TempO-STAT assay, and the assay itself, was validated using white cell biomarkers of radiation exposure and human cell lines. Biomarkers for CLas infection of psyllids and citrus were identified, reference RNA from uninfected and infected psyllid and leaves has been prepared and measured by the benchmark qPCR assay by Dr. Brown, and these samples are being tested in the TempO-Seq assay. The TempO-Seq measurement of differentially expressed genes correlates to the benchmark qPCR data. The psyllid assay was pursued. Research Findings - CLas biomarkers for psyllids and citrus were identified and put into the TempO-Seq assay. Reference RNA from infected psyllids and citrus leaves was obtained and validated using the benchmark qPCR assay in Dr. Brown's lab. The TempO-Seq measurement of differentially expressed genes from this reference RNA correlates to the benchmark qPCR data. Potential Applications - The expected outcomes of this program are thedemonstration ofthe feasibility of a multiplexed qPCR-like assay and aTempO-Seq targeted sequencing assay to identify CLas infection in psyllids (transmitting insects) and citrus leaves.Both tests will be used to identify CLas infection in the insects that transmit citrus greening and in the trees themselves, to provide a basis for identifying infected tress and preventing infection from reaching uninfected trees. Accomplishments vs Goals and Milestones: Aim 1: Biomarker selection for the TempO-STAT and TempO-Seq assays and validation in the TempO-Seq assay. Success Criteria: Content suitable for the TempO-STAT assay identified and validated based on sequencing data from the TempO-Seq assay of reference psyllid and tree samples correlating to q-PCR CLas data for those genes measured by the more limited content qPCR benchmark assay (R2>0.7 cross-platform correlation). Status: completed. Focus was on the Psyllid assay. RNA was extracted from infected reference psyllid samples obtained from Florida (after quarantine), tested in the benchmark qPCR assay in the laboratory of Dr. Brown, and tested using the TempO-Seq assay to validate the detector oligos (which will also be used in the TempO-STAT assay) and to identify the genes that will be measured in the TempO-STAT assay. Content for the TempO-STAT assay was selected, and the DOs and labeled probes to measure each ordered: DO IDT plate Position Probe Fluorophor PSYL_44128 20171212_WolbPsylClib B06 Hex CLIBASIA_02490 20171212_WolbPsylClib B07 FAM PSYL_38381 20171212_WolbPsylClib C05 Hex PSYL_03404 20171212_WolbPsylClib D02 Hex CLIBASIA_02610 20171212_WolbPsylClib E07 Cy 5 PSYL_07787 20171212_WolbPsylClib G02 Hex CLIBASIA_02170 20171212_WolbPsylClib G06 FAM CLIBASIA_02850 20171212_WolbPsylClib G07 Cy 5 PSYL_09963 20171212_WolbPsylClib H02 Hex CLIBASIA_02905 20171212_WolbPsylClib H07 FAM CLIBASIA_00140 20180118_PhageClibasia D02 FAM CLIBASIA_00825 20180118_PhageClibasia F02 FAM
Publications
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