Jisheng Li, Kelly Li, Nicole Fantin, Sunali Patel, Bonnie Moy, Toinette Hartshorne, Boli Huang; Hong Ji, Ioanna Pagani, Emily Zeringer, Nitin Puri, and Kamini VarmaClinical 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 of the leading causes of death globally, respiratory infections could be caused by s ingle or multiple types of v iral, bacterial or fungal pathogens that present in the upper and lower respiratory tract. Panel-based testing using molecular methods to identify multiple pathogens simultaneously can contribute to better understanding of respiratory infections.

MATERIALS AND METHODS

To develop a comprehensive and flexible research panel, we chose TaqMan® OpenArrayTMplatform to identify common respiratory tract organisms via real-time PCR technology. Target organism sequences were acquired from IMG and NCBI database. Divergent gene targets were chosen to design research assays with high specific ity (ANI >96%) and wide strain coverage. For each target, multiple assays were designed in s ilico and then assay performance was evaluated using various controls including synthetic and natural genomic DNA and RNA, as well as human respiratory specimen. Assay specific ity was evaluated with genomic RNA and DNA of standard reference viral and bacterial organisms from American Type Culture Collection (ATCC), To achieve higher sensitiv ity, optimized components and thermal cycling condition for PCR pre-amplification was determined by extensive Design of Experiments (DOE) studies.

When tested with genomic RNA and DNA of standard reference viral and bacterial organisms from American Type Culture Collection (ATCC), the assays in RTM panel displayed high specific ity. All organisms are detected by relevant assays .

Plots show Crtvalues of selected assays at different input (copies/µL) of synthetic targets. The resulted Ct values show linear response to the copy number of targets, demonstrating high PCR effic iency, which confirms in s ilico design prediction.

Charts show Ct values (Y-axes) corresponding to different amount of initial input copies (X-axes, at power of 10 copies/µL) of ATCC viral and bacterial controls.

OpenArrayTMtechnology utilizes a microscope slide–sized plate with 3,072 through-holes. Each plate contains 48 subarrays with 64 through-holes. Assays for different RTM targets are loaded in indiv idual through-holes. 1-4 subarrays can form a RTM panel depending on customer specification of the number of targets and replicates. 12 – 48 samples may be processed simultaneously on one OpenArray plate for RTM studies.

Sample Prep

ü Nasophar yngeal Swab

ü Nasophar yngeal Aspir at e

ü Br onchoalveolar Lavage

RT Pr e-

am plif icat ion

Pla te loading

and qPCRAccuFillT M

Q uant St udioT M

Data Analysis

DNA and RNA from various types of samples of respiratory tract may be extracted with KingFisherTMFlex purification system, followed by reverse transcription and pre-amplification. The reaction is then diluted and loaded into OpenArrayTMplates with AccuFillTMsystem. Real time PCR of RTM assays is performed in QuantStudioTM12K Flex instrument and results of positive or negative are analyzed with QuantStudioTM12 Flex software.

RESULTS

ID037

Assays/Controls

Hum

anade

novirus2

Hum

anade

novirus7

Hum

anade

novirus12

Bordetellabronchisepe

tica

Bordetellaparap

ertussis

Bordetellaholmesii

Bordetellapertussis

Chlamyd

ophila

pneu

mon

iae

Chlamyd

ophila

pneu

mon

iae

Hum

ancoron

avirus

229E

Betacorona

virus1

strainOC4

3Hum

anHerpe

svirus5

HCM

V

EnterovirusD68

(US/KY

/14-18953)

EnterovirusD68

(US/MO/14-18947)

Haemop

hilusinflu

enzae

Influ

enzaAvirus(H

1N1)

strainA/

Influ

enzaAvirus(H

3N2)

Influ

enzaBvirus

Kieb

siellapne

umon

iae

Legion

ellapne

umop

hila

Measlesvirus

Moraxellacatarrhalis

Mum

psvirus

Mycop

lasm

apn

eumon

iae

Parainflu

enzaPIV-1

Parainflu

enzaPIV-2

Parainflu

enzaPIV-3

Parainflu

enzaPIV-4

Hum

anrespiratorySyn

cytial

Virus

Hum

anrespiratorySyn

cytial

Virus

Hum

anrhino

virus17

Stap

hylococcusaureu

s

Streptococcuspn

eumon

iae

Hum

anherpe

svirus3

Adenovirus_pan_1 21.8 20.4Adenovirus_pan_2 20.7 21.0Bordetella_pan 23.9 22.7 22.4B_holmesii 22.6B_pertussis 22.6C_pneumoniae 22.3 20.2Coronavirus_229E 22.6Coronavirus_OC43 21.7HHV5_CMV 20.1Enterovirus_D68 23.6 23.6Enterovirus_pan 28.4H_influenzae 19.7Influenza_A_H1 23.2Influenza_A_pan 20.7 22.6Influenza_B 21.6K_pneumoniae 20.1L_pneumophila 20.8Measles 21.6M_catarrhalis 20.9Mumps 23.0M_pneumoniae 20.7PIV_1 22.0PIV_2 22.1PIV_3 21.4PIV_4 20.4RSVA 20.0RSVB 28.5 21.8Rhinovirus_pan_1 25.5S_aureus 20.1S_pneumoniae 19.0HHV3_VZV 20.7

A novel research application has been developed for Respiratory Tract Microbiota (RTM) profiling. Assays in the panel demonstrate desirable performance in terms of sensitiv ity, specific ity and linearity range. The application enables both customizable and high-throughput panels for respiratory infection research and provides a cost-effective tool for researchers to understand pathogenic ity in respiratory tract infections.

Genomic materials from ATCC were pooled and titrated at serial dilutions for the tests. The left panel shows Ct values of pools when applied for real-time PCR test on OpenArrayTMdirectly after reverse transcription. The right panel shows Ct values of corresponding pools underwent a pre-amplification process at which 14 cycles of polymerase chain reaction was carried out following reverse transcription.

0

5

10

15

20

25

30

35

CtValue

q P CR withou tP re-amp l i fi cation

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

CtValue

q P CR with P re-amp l i fi cation

PA_10^0 PA_10^1 PA_10^2 PA_10^3 PA_10^4 PA_10^5

CONCLUSIONS

For Research Use Only. Not for use in diagnostic procedures.

© 2018 Thermo Fisher Scientific Inc. All rights reserved.

All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan® is a registered trademark of Roche 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 workflowFigure 5. Linearity range of RTM assays

Figure 1. OpenArrayTM system for high throughput RTM profiling.

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