TITLE: Borrelia burgdorferi ospC Genotyping Using Luminex Technology
AUTHORS: Patrick  Pearson, Olivia Skaltsis, Chu-Yuan Luo, Guang Xu, Zachary Oppler, Dustin Brisson, Stephen M Rich

[DESCRIPTION]
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.

[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. 

[STEPS]
SECTION: PREPARING SOLUTIONS
1. 1X xTAG buffer

Add 1 mL of 10X xTAG buffer to 9 mL of molecular grade water. Scale volume up or down as necessary.
Store at 4°C until use
SECTION: PREPARING SOLUTIONS
2. 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.

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

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

 
     Name       Sequence (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 (#2)      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   
 
SECTION: PREPARING SOLUTIONS
4. 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

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

SECTION: NESTED PCR
6. 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

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

 

     Component      Volume (ul)        Promega PCR Master Mix (2X)      12.5        ospC1F (10 uM)      0.5        ospC622RC (10 uM)      0.5        Molecular grade water      10.5   
 
Loading master mix and template DNA

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

SECTION: NESTED PCR
7.  
Nested PCR Program

     Temperature (°C)      Time       Cycles        95      2 minutes      1        95      30 seconds      32        49      30 seconds        72      50 seconds        72      10 minutes      1        4      Hold      1   

SECTION: NESTED PCR
8. Second Round PCR

Master mix preparation

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

 

 
     Component      Volume (ul)        Promega PCR Master Mix (2X)      12.5        OC6 (+) 24 (10 uM)      0.5        OC602 (-) 22 (10 uM)      0.5        Molecular grade water      10.0   
 
Loading master mix and template DNA

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


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


 

SECTION: ALLELE SPECIFIC PRIMER EXTENSION (ASPE)
10. Master mix preparation

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


 

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

 

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


 

SECTION: ANALYSIS ON MAGPIX
12. Addition of SAPE (reporter solution)

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

In a 15 mL tube, aliquot 87 μL SAPE into 8613 μL 1X xTAG buffer
Total volume is 8700 μL 
Make fresh immediately before use in step 12 below
SECTION: ANALYSIS ON MAGPIX
13. 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.

 70 μL sample volume
 Sample wash "on"
 Heater set at 37°C
 50 bead count minimum
SECTION: DATA ANALYSIS
14. 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

 Open the .csv file in Microsoft Excel
 Copy the "DataType: Median" results for the three NTCs and every sample to a new sheet in the workbook
 Using the three NTCs, calculate the average background MFI value for every ospC genotype (analyte)
 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.
 For individual samples, raw ospC genotype MFI values that have a ratio to NTC value (RNTC) of ≥ 3 are considered positive

Example

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