Sep 01, 2023

Borrelia burgdorferi ospC Genotyping Using Luminex Technology V.2

PLOS One
Peer-reviewed method
DOI
https://dx.doi.org/10.17504/protocols.io.5jyl899k8v2w/v2
  • Patrick Pearson1,
  • Olivia Skaltsis2,
  • Chu-Yuan Luo1,
  • Guang Xu1,
  • Zachary Oppler3,
  • Dustin Brisson3,
  • Stephen M Rich1
  • 1Laboratory of Medical Zoology, Department of Microbiology, University of Massachusetts, Amherst, Massachusetts, United States of America;
  • 2NH Citizens Health Initiative, Institute for Health Policy and Practice, University of New Hampshire, Concord, New Hampshire, United States of America;
  • 3Evolution and Ecology Disease Systems Laboratory, Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America
  • PLOS ONE Lab Protocols
  • Spotlight series
Patrick Pearson
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DOI: https://dx.doi.org/10.17504/protocols.io.5jyl899k8v2w/v2
External link: https://doi.org/10.1371/journal.pone.0269266
Protocol Citation: Patrick Pearson, Olivia Skaltsis, Chu-Yuan Luo, Guang Xu, Zachary Oppler, Dustin Brisson, Stephen M Rich 2023. Borrelia burgdorferi ospC Genotyping Using Luminex Technology. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl899k8v2w/v2Version created by Patrick Pearson
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 01, 2023
Last Modified: September 01, 2023
Protocol Integer ID: 87219
Keywords: Borrelia burgdorferi, ospC, genotyping, using luminex technology borrelia burgdorferi, luminex technology borrelia burgdorferi, borrelia burgdorferi ospc, burgdorferi ospc, important tickborne human pathogen, luminex xmap technology for the detection, infected tick, multiplex assay, luminex xmap technology, ospc gene, borrelia, characterization of different ospc genotype, characterization of ospc genotype, ospc genotype, hybridization to luminex microsphere, different ospc genotype, luminex microsphere, amplification of the ospc gene, assay, borrelia burgdorferi ospc genotyping
Abstract
Borrelia burgdorferi is an important tickborne human pathogen and can be grouped into separate strains based on the outer surface protein C (ospC) gene. The detection and characterization of different ospC genotypes is vital for research on B. burgdorferi and the risk it poses to humans. Here we present a novel, multiplex assay based on Luminex xMAP technology for the detection of B. burgdorferi ospC genotypes. The assay has 5 major steps: amplification of the ospC gene, enzymatic purification, incorporation of biotinylated nucleotides into the template DNA, hybridization to Luminex microspheres, and detection of fluorescent signals corresponding to each ospC genotype. This protocol can be used for the characterization of ospC genotypes in B. burgdorferi infected ticks, reservoir hosts, and/or clinical samples.

(The last step in this version contains a supplemental video with extra context and tips, as part of the protocols.io Spotlight series, featuring conversations with protocol authors.)
Guidelines
The Luminex xMAP Cookbook is available on the Luminex website and helpful for designing and troubleshooting genotyping assays.

The nucleic acid extraction depends on the type of sample being tested. We recommend using MasterPure™ Complete DNA and RNA Purification Kit (Lucigen) and following their protocols.

Positive controls are heavily recommended. One approach to generate positive controls is to use synthetic, double-stranded gene fragments (gBlocks) from IDT, corresponding to each ospC genotype. Amplify the gBlocks and mix into pools of positive controls accounting for every ospC genotype.
Materials
Reagents and Consumables
MaterialVendor (Catalog Number)
PCR PlateFisher (E951020460)
8 Strip Flat CapsFisher (E0030124847)
Molecular Grade WaterFisher (BP281910)
PCR 2X Master MixPromega (M7832)
Nested PCR and ASPE PrimersIDT
ExoSAP-ITThermoFisher (78201.1.ML)
dNTP Solution SetNEB (N0446S)
Biotin-14-dATPThermoFisher (19524016)
NEB Taq Polymerase wih 10X BufferNEB (M0273S)
Streptavidin, R-Phycoerythrin Conjugate (SAPE)Thermofisher (S-866)
xTAG 10X BufferLuminex (GR001C0060)
Drive FluidLuminex (40-50030)
Calibration KitLuminex (MPX-CAL-K25)
Performance Verification KitLuminex (MPX-PVER-K25)
MicrospheresLuminex
25 mL ReservoirFisher (14387070
Amber 5 mL TubeAxygen (MCT-500-X)
2 mL TubeEppendorf (022363352)
15 mL TubeEppendorf (0030122151)

Luminex Microspheres
Bead RegionCatalog NumberAnti-TAG Sequence Bound to Each Microsphere (5'-3')Complementary Primer TAG Sequence (5'-3')Matching ospC Genotype
12 MTAG-A012 AGTAGAAAGTTGAAATTGATTATG CATAATCAATTTCAACTTTCTACT All
14 MTAG-A014 ATTGTGAAAGAAAGAGAAGAAATT AATTTCTTCTCTTTCTTTCACAAT A
15 MTAG-A015 GTTGTAAATTGTAGTAAAGAAGTA TACTTCTTTACTACAATTTACAAC B
18 MTAG-A018 GTAATTGAATTGAAAGATAAGTGT ACACTTATCTTTCAATTCAATTAC D
20 MTAG-A020 AAATTAGTTGAAAGTATGAGAAAG CTTTCTCATACTTTCAACTAATTT K
22 MTAG-A022 GATTGATATTTGAATGTTTGTTTG CAAACAAACATTCAAATATCAATC I/C
30 MTAG-A030 GTGTTATAGAAGTTAAATGTTAAG CTTAACATTTAACTTCTATAACAC M
36 MTAG-A036 TTGTGTAGTTAAGAGTTGTTTAAT ATTAAACAACTCTTAACTACACAA E
38 MTAG-A038 AGTAAGTGTTAGATAGTATTGAAT ATTCAATACTATCTAACACTTACT T
42 MTAG-A042 ATTTGTTATGATAAATGTGTAGTG CACTACACATTTATCATAACAAAT F
44 MTAG-A044 AATGTAAAGTAAAGAAAGTGATGA TCATCACTTTCTTTACTTTACATT G
46 MTAG-A046 GTGATTGAATAGTAGATTGTTTAA TTAAACAATCTACTATTCAATCAC H
48 MTAG-A048 TATGAATGTTATTGTGTGTTGATT AATCAACACACAATAACATTCATA I
52 MTAG-A052 GTAAGATTAGAAGTTAATGAAGAA TTCTTCATTAACTTCTAATCTTAC J
54 MTAG-A054 TAGAGAAAGAGAGAATTGTATTAA TTAATACAATTCTCTCTTTCTCTA L
56 MTAG-A056 AATTAGAAGTAAGTAGAGTTTAAG CTTAAACTCTACTTACTTCTAATT N
61 MTAG-A061 TATTAGAGAGAAATTGTAGAGATT AATCTCTACAATTTCTCTCTAATA O
63 MTAG-A063 TTTGTTGTTAAGTATGTGATTTAG CTAAATCACATACTTAACAACAAA U
65 MTAG-A065 TGAGTAAGTTTGTATGTTTAAGTA TACTTAAACATACAAACTTACTCA V
67 MTAG-A067 TTTGTGTGTTATTGTAATTGAGAT ATCTCAATTACAATAACACACAAA W
72 MTAG-A072 AATTGAGAAAGAGATAAATGATAG CTATCATTTATCTCTTTCTCAATT E/C
Table 1: Luminex microspheres (beads)

Equipment
  1. Thermal cycler
  2. Luminex MAGPIX instrument
  3. 96-well plate centrifuge
  4. Multichannel pipettes

PREPARING SOLUTIONS
1X xTAG buffer

  1. Add 1 mL of 10X xTAG buffer to 9 mL of molecular grade water. Scale volume up or down as necessary.
  2. Store at 4°C until use
Bead mix solution (75 beads/μL)

The specific Luminex microspheres (beads) are sold in concentrations of 2.5X10^6 beads/mL. The final concentration of a working bead mix solution is 75 beads/μL. For a 96 well plate, make enough bead mix for 115 samples (~1.2X) to account for pipetting error. Scale volume up or down as necessary.

  1. Vortex each bead type (Table 1 from Materials) for at least 20 seconds
  2. Add 69 μL of each bead type to an amber 5 mL tube
  3. The volume with 21 bead types will be 1449 μL
  4. Add 851 μL of 1X xTAG buffer to bring total volume to 2300 μL
  5. Store at 4°C until use

ASPE primer mix solution (500 nM)
  1. Dilute ASPE primers (from IDT) to 200 uM
  2. In 2 mL tube, add 947.5 μL of molecular grade water
  3. Add 2.5 μL of each ASPE primer (Table 2 below)
  4. Vortex and store at -20°C until use

PrimerSequence (5'-3')
ospC ALL Tag 12 CATAATCAATTTCAACTTTCTACTAGATTAGGCCCTTTAACAGACTCATC
ospC Type A Tag 14 AATTTCTTCTCTTTCTTTCACAATATTGTGATTATTTTCGGTATCC
ospC Type B Tag 15 TACTTCTTTACTACAATTTACAACCTCGTTGCGATTTGCTTCA
ospC Types E/C Tag 72 CTATCATTTATCTCTTTCTCAATTTGCAAGTAAGGTCTCAACTT
ospC Types I/C Tag 22 CAAACAAACATTCAAATATCAATCTCCGTTGTTATCTGCCTCATTATCT
ospC Type D Tag 18 ACACTTATCTTTCAATTCAATTACATGATTATTTAGAGTGCCTAAAGCATTGTTTTGATC
ospC Type E Tag 36 ATTAAACAACTCTTAACTACACAATGTGTTTTTACTCTGATTGGCCTCTAAACCATTATTGCC
ospC Type F Tag 42 CACTACACATTTATCATAACAAATCGCCTGAACGCCTAAACCATTTGCATC
ospC Type G Tag 44 TCATCACTTTCTTTACTTTACATTGGTGTTGTGATTCGCATCAG
ospC Type H Tag 46 TTAAACAATCTACTATTCAATCACGCCCCCATCGTCACCCAAAGTGCCATTTTG
ospC Type I Tag 48 AATCAACACACAATAACATTCATATTTGAAATTAAATATGCTCCTGA
ospC Type J Tag 52 TTCTTCATTAACTTCTAATCTTACTCCGTTTTGACCCACTTCAGC
ospC Type K Tag 20 CTTTCTCATACTTTCAACTAATTTCCCCGCTTCGACAGCTAAACCACCATTTTGTTG
ospC Type L Tag 54 TTAATACAATTCTCTCTTTCTCTAATCGCTACCTAAAGTACCACCTGCTTC
ospC Type M Tag 30 CTTAACATTTAACTTCTATAACACACCGGCATTTAAACCATTTTGGGCTATCAAA
ospC Type N Tag 56 CTTAAACTCTACTTACTTCTAATTGTTTTGCACATCATCTAAACCATTATTATT
ospC Type O Tag 61 AATCTCTACAATTTCTCTCTAATATTGGTTAACTAAGCCATTTGCC
ospC Type T Tag 38 ATTCAATACTATCTAACACTTACTATGGCCTGCATCGACACT
ospC Type U Tag 63 CTAAATCACATACTTAACAACAAACTGCCCTTGCAAGTCCTGT
ospC Type V Tag 65 TACTTAAACATACAAACTTACTCAGAGCCGCTTGAGCAGTTAAACCATTTGCACC
ospC Type W Tag 67 ATCTCAATTACAATAACACACAAATCGTTTCGATTTGCTTCTACACCC
Table 2: ASPE Primers
ASPE dNTP Mix (50 μM)

Before making this solution, dilute the dTTP, dCTP, and dGTP (NEB) from 100 mM to 10 mM, and discard the dATP

  1. In 2 mL tube, add 430 μL of molecular grade water
  2. Add 2.5 μL of each 10 mM dTTP, dCTP, and dGTP
  3. Add 62.5 μL of 0.4 mM biotin-14-dATP
  4. Vortex and store at -20°C until use

Streptavidin, R-phycoerythrin conjugate (SAPE) solution

The SAPE is supplied as 1 mg/mL solution. Dilute SAPE to 10 μg/mL in 1X xTAG buffer. For a 96 well plate, make enough SAPE solution for 116 samples (~1.2X). Scale up or down as necessary.

  1. In a 15 mL tube, aliquot 87 μL SAPE into 8613 μL 1X xTAG buffer
  2. Total volume is 8700 μL
  3. Make fresh immediately before use in step 12 below
NESTED PCR
Note: Every batch of samples must have three no template controls (NTCs) included at the nested PCR step. They are used to determine positive ospC genotypes after analysis on the MAGPIX instrument, and to confirm that no contamination occurred during the assay.

First round PCR

Master mix preparation

  1. Prepare master mix according to Table 4 below for each sample. Scale up depending on the sample size
  2. Add master mix components to a tube, vortex, and store on ice

AB
PrimerSequence (5'-3')
ospC1FATGAAAAAGAATACATTAAGTGCA
ospC622RCTTGGACTTTCTGCCACAACA
Table 3: First round nested PCR primers

AB
ComponentVolume (μL)
Promega PCR Master Mix (2X) 12.5
ospC1F (10 uM) 0.5
ospC622RC (10 uM) 0.5
Molecular grade water 10.5
Table 4: First round PCR master mix
Loading master mix and template DNA

  1. Aliquot 24 μL of the master mix into a well for each sample
  2. Aliquot 1 μL of template DNA or NTC
  3. Seal wells firmly with cap strips
  4. Vortex and centrifuge plate at 2500 RPM for 1 minute
  5. Load plate into thermal cycler and run program in Table 5 (step 7)

Nested PCR Program

ABC
Temperature (°C) Time Cycles
95 2 minutes 1
95 30 seconds 32
49 30 seconds 32
72 50 seconds 32
72 10 minutes 1
4 Hold 1
Table 5: Nested PCR program

Second Round PCR

Master mix preparation

  1. Prepare master mix according to Table 7 below for each sample. Scale up depending on the sample size
  2. Add master mix components to a tube, vortex, and store on ice

AB
PrimerSequence (5'-3')
OC6(+)24AAAGAATACATTAAGTGCGATATT
OC602(-)22GGGCTTGTAAGCTCTTTAACTG
Table 6: Second round nested PCR primers

AB
Component Volume μL
Promega PCR Master Mix (2X) 12.5
OC6(+)24 (10 μM) 0.5
OC602(-)22 (10 μM) 0.5
Molecular grade water 10.0
Table 7: Second round PCR master mix
Loading master mix and template DNA

  1. Aliquot 23.5 μL of the master mix into a well for each sample
  2. Add 1.5 μL of the DNA template or NTC from the first round PCR into the correct well
  3. Seal wells firmly with cap strips
  4. Vortex and centrifuge plate at 2500 RPM for 1 minute
  5. Load plate into thermal cycler and run program in Table 5 (step 7)


EXOSAP-IT
  1. Aliquot 7.5 μL of amplified DNA into wells on a new plate
  2. Add 3 μL of ExoSAP-IT to each well
  3. Seal wells firmly with cap strips
  4. Vortex and centrifuge plate at 2500 RPM for 1 minute
  5. Load plate into thermal cycler and run program in Table 8


ABC
Temperature (°C) Time (minutes)Cycles
37151
80151
4Hold1
Table 8: ExoSAP-IT program

ALLELE SPECIFIC PRIMER EXTENSION (ASPE)
Master mix preparation

  1. Prepare master mix according to Table 9 below for each sample. Scale up depending on the sample size
  2. Add master mix components to a tube, vortex, and store on ice


AB
ComponentVolume (μL)
Molecular grade water9.75
10X NEB PCR buffer2
ASPE primer mix (500 nM)2
ASPE dNTP mix (50 μM)1
NEB Taq polymerase (5 U/μL)0.25
Table 9: ASPE master mix

  1. Aliquot 15 μL of the ASPE master mix into wells on a new plate
  2. Aliquot 5 μL of the amplicon treated product (from step 9) into the correct wells
  3. Seal wells firmly with cap strips
  4. Vortex and centrifuge plate at 2500 RPM for 1 minute
  5. Load plate into thermal cycler and run program in Table 10

ABC
Temperature (°C) Time Cycles
952 minutes1
9530 seconds35
5630 seconds35
6830 seconds35
68 5 minutes1
4Hold1
Table 10: ASPE program

BEAD HYBRIDIZATION
  1. Vortex bead mix for at least 30 seconds
  2. Aliquot 20 μL of bead mix into wells on a new plate
  3. Aliquot 3.5 μL of the ASPE product (from step 10) into the correct wells
  4. Gently pipette up and down several times to mix
  5. Seal wells firmly with cap strips
  6. Load plate into thermal cycler and run program in Table 11


ABC
Temperature (°C) Time (minutes)Cycles
9621
37301
Table 11: Bead hybridization program

ANALYSIS ON MAGPIX
Addition of SAPE (reporter solution)

  1. Invert SAPE (10 μg/mL) several times and pour into 25 mL reservoir
  2. Aliquot 75 μL SAPE solution to every well
  3. Gently pipette up and down several times to mix
  4. Transfer samples to pre-warmed heater block (37°C) on MAGPIX instrument
  5. Incubate at 37°C for 15 minutes
Analyze samples on MAGPIX

Please refer to Luminex technical support and the MAGPIX manual for detailed instructions on how to create a protocol, run a batch of samples, and MAGPIX maintenance. Specific settings for our ospC genotyping protocol are listed below.

  1. 70 μL sample volume
  2. Sample wash "on"
  3. Heater set at 37°C
  4. 50 bead count minimum
DATA ANALYSIS
At the end of the run, a .csv file will be created containing the protocol/run information and the raw median fluorescent intensity (MFI) results for each sample

  1. Open the .csv file in Microsoft Excel
  2. Copy the "DataType: Median" results for the three NTCs and every sample to a new sheet in the workbook
  3. Using the three NTCs, calculate the average background MFI value for every ospC genotype (analyte)
  4. Add three standard deviations to the averages. These values (NTC values) are specific for each genotype. NTC values are determined for each genotype, since background can vary slightly between genotypes.
  5. For individual samples, raw ospC genotype MFI values that have a ratio to NTC value (RNTC) of ≥ 3 are considered positive

Example

  1. For ospC genotype A the MFI values in the three NTCs were (100, 125, and 150)
  2. The average (125) plus three standard deviations (20.4) is 186 (NTC value)
  3. The raw MFI value for ospC genotype A in sample "X" is 800
  4. Sample "X" is positive for ospC genotype A since the RNTC value is 4.3

Spotlight video