A High Throughput System for Profiling Respiratory Tract Microbiota
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A panel was developed using the OpenArray platform to profile common respiratory tract pathogens via PCR. Assays were designed to target viral and bacterial sequences with high specificity and strain coverage. The panel demonstrated high specificity when tested against genomic standards. Pre-amplification improved sensitivity by enhancing detection of low copy targets. The panel provides a customizable and high-throughput tool for respiratory infection research.
A High Throughput System for Profiling Respiratory Tract Microbiota
- 1. Jisheng Li, Kelly Li, Nicole Fantin, Sunali Patel, Bonnie Moy, Toinette Hartshorne, Boli Huang; Hong Ji, Ioanna Pagani, Emily Zeringer, Nitin Puri, and Kamini Varma Clinical R&D, Genetic Science Division, Life Science Group, Thermo Fisher Scientific, 180 Oyster Point Blvd, South San Francisco, CA 94080 A High Throughput System for Profiling Respiratory Tract Microbiota INTRODUCTION As one ofthe leading causes ofdeath globally,respiratory infections could be caused bysingleor multiple typesofviral, bacterial or fungal pathogensthatpresentin the upper and lower respiratorytract.Panel-basedtesting using molecular methods to identify multiple pathogens simultaneouslycan contribute to better understanding ofrespiratoryinfections. MATERIALS AND METHODS To develop a comprehensiveand flexibleresearch panel,we chose TaqMan® OpenArrayTM platform to identify common respiratory tractorganisms via real-time PCR technology.Target organism sequenceswere acquiredfrom IMG and NCBI database.Divergentgene targetswerechosento design research assays with highspecificity (ANI>96%) and wide strain coverage.For each target,multiple assays were designed in silico and then assay performance was evaluated using various controlsincluding synthetic and natural genomic DNAand RNA,as well as human respiratoryspecimen. Assay specificity was evaluated with genomic RNAand DNAof standard reference viral and bacterial organisms from American Type Culture Collection (ATCC), To achieve higher sensitivity, optimized components andthermal cyclingcondition for PCR pre-amplification was determined byextensiveDesign of Experiments (DOE) studies. When tested with genomic RNAandDNAofstandard reference viral and bacterial organismsfrom AmericanTypeCultureCollection (ATCC),the assays in RTM panel displayed highspecificity. All organisms are detected by relevantassays. Plots show Crtvalues ofselected assays atdifferentinput(copies/µL) ofsynthetic targets. The resulted Ctvalues show linear response to the copy number oftargets, demonstrating high PCR efficiency,which confirms in silico design prediction. Charts show Ctvalues (Y-axes)corresponding to differentamountofinitial inputcopies (X-axes,atpower of10 copies/µL) ofATCC viral andbacterial controls. OpenArrayTM technologyutilizes a microscope slide–sized plate with 3,072 through-holes.Eachplate contains48 subarrays with 64 through-holes.Assaysfor differentRTM targets are loaded in individual through-holes.1-4subarrayscan form a RTM panel depending on customerspecificationofthe number oftargets and replicates. 12 – 48 samples may beprocessedsimultaneously on one OpenArray plate for RTM studies. Sample Prepü Nasophar yngeal Swab ü Nasophar yngeal Aspir at e ü Br onchoalveolar Lavage RTPr e- am plif icat ion Plate loading and qPCR AccuFill T M Q uant St udio T M Data Analysis DNAand RNAfrom varioustypesofsamplesofrespiratorytractmay be extracted with KingFisherTM Flex purification system,followed byreverse transcription and pre-amplification. The reaction is thendiluted and loaded into OpenArrayTM plates with AccuFillTM system. Real time PCR ofRTM assaysis performed in QuantStudioTM 12KFlex instrumentand results ofpositiveor negative are analyzed with QuantStudioTM 12 Flex software. RESULTS ID037 Assays/Controls Human adenovirus 2 Human adenovirus 7 Human adenovirus 12 Bordetella bronchisepetica Bordetella parapertussis Bordetella holmesii Bordetella pertussis Chlamydophila pneumoniae Chlamydophila pneumoniae Human coronavirus 229E Betacoronavirus 1 strain OC43 Human Herpes virus 5 HCMV Enterovirus D68 (US/KY/14-18953) Enterovirus D68 (US/MO/14-18947) Haemophilus influenzae Influenza A virus (H1N1) strain A/ Influenza A virus (H3N2) Influenza B virus Kiebsiella pneumoniae Legionella pneumophila Measles virus Moraxella catarrhalis Mumps virus Mycoplasma pneumoniae Parainfluenza PIV-1 Parainfluenza PIV-2 Parainfluenza PIV-3 Parainfluenza PIV-4 Human respiratory Syncytial Virus Human respiratory Syncytial Virus Human rhinovirus 17 Staphylococcus aureus Streptococcus pneumoniae Human herpesvirus 3 Adenovirus_pan_1 21.8 20.4 Adenovirus_pan_2 20.7 21.0 Bordetella_pan 23.9 22.7 22.4 B_holmesii 22.6 B_pertussis 22.6 C_pneumoniae 22.3 20.2 Coronavirus_229E 22.6 Coronavirus_OC43 21.7 HHV5_CMV 20.1 Enterovirus_D68 23.6 23.6 Enterovirus_pan 28.4 H_influenzae 19.7 Influenza_A_H1 23.2 Influenza_A_pan 20.7 22.6 Influenza_B 21.6 K_ pneumoniae 20.1 L_pneumophila 20.8 Measles 21.6 M_catarrhalis 20.9 Mumps 23.0 M_pneumoniae 20.7 PIV_1 22.0 PIV_2 22.1 PIV_3 21.4 PIV_4 20.4 RSVA 20.0 RSVB 28.5 21.8 Rhinovirus_pan_1 25.5 S_aureus 20.1 S_pneumoniae 19.0 HHV3_VZV 20.7 Anovel researchapplication hasbeen developed for Respiratory TractMicrobiota (RTM) profiling.Assays in the panel demonstrate desirable performance in termsofsensitivity,specificity and linearity range.The application enables both customizable and high- throughputpanels for respiratory infection research andprovidesa cost-effective tool for researchersto understand pathogenicity in respiratory tractinfections. Genomic materials from ATCC were pooled and titrated atserial dilutions for the tests. The leftpanel shows Ctvaluesofpools when applied for real-time PCR teston OpenArrayTM directly after reversetranscription. The rightpanel shows Ctvalues ofcorrespondingpools underwenta pre- amplification process atwhich 14 cycles ofpolymerase chain reaction was carried outfollowing reversetranscription. 0 5 10 15 20 25 30 35 Ct Value qPCR without Pre-amplification RT_10^0 RT_10^1 RT_10^2 RT_10^3 RT_10^4 RT_10^5 0. 0 5. 0 10. 0 15. 0 20. 0 25. 0 30. 0 35. 0 Ct Value qPCR with Pre-amplification PA_10^0 PA_10^1 PA_10^2 PA_10^3 PA_10^4 PA_10^5 CONCLUSIONS For Research Use Only.Notfor usein diagnostic procedures. © 2018 Thermo Fisher Scientific Inc.All rights reserved. All trademarks are the property ofThermo Fisher Scientific and its subsidiaries unlessotherwise specified.TaqMan® is a registered trademark ofRoche Molecular Systems,Inc.,used under permission and license. Table 2. Specificity of OpenArrayTM Respiratory Tract Microbiota (RTM) Panel Figure 4. Pre-amplification enhances assay sensitivity Figure 3. qPCR results of Thermo Fisher Scientific Respiratory Tract Microbiota (RTM) panel assays tested with synthetic assay targets (SP) at serial dilutions. Table 1. List of targeted organisms of Thermo Fisher Scientific OpenArrayTM Respiratory Tract Microbiota (RTM) research panel Figure 2. RTM study workflow Figure 5. Linearity range of RTM assays Figure 1. OpenArrayTM system for high throughput RTM profiling. Thermo Fisher Scientific •5781 Van Allen Way •Carlsbad, CA92008 •thermofisher.com