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

A	B
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

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

A	B
Primer	Sequence (5'-3')
ospC1F	ATGAAAAAGAATACATTAAGTGCA
ospC622RC	TTGGACTTTCTGCCACAACA

Table 3: First round nested PCR primers

A	B
Component	Volume (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

A	B	C
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

A	B
Primer	Sequence (5'-3')
OC6(+)24	AAAGAATACATTAAGTGCGATATT
OC602(-)22	GGGCTTGTAAGCTCTTTAACTG

Table 6: Second round nested PCR primers

A	B
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)

Aliquot 7.5 μL of amplified DNA into wells on a new plate1. Add 3 μL of ExoSAP-IT to each well

1. Seal wells firmly with cap strips
2. Vortex and centrifuge plate at 2500 RPM for 1 minute
3. Load plate into thermal cycler and run program in Table 8

A	B	C
Temperature (°C)	Time (minutes)	Cycles
37	15	1
80	15	1
4	Hold	1

Table 8: ExoSAP-IT program

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

A	B
Component	Volume (μL)
Molecular grade water	9.75
10X NEB PCR buffer	2
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

A	B	C
Temperature (°C)	Time	Cycles
95	2 minutes	1
95	30 seconds	35
56	30 seconds	35
68	30 seconds	35
68	5 minutes	1
4	Hold	1

Table 10: ASPE program

Vortex bead mix for at least 30 seconds1. Aliquot 20 μL of bead mix into wells on a new plate

1. Aliquot 3.5 μL of the ASPE product (from step 10) into the correct wells
2. Gently pipette up and down several times to mix
3. Seal wells firmly with cap strips
4. Load plate into thermal cycler and run program in Table 11

A	B	C
Temperature (°C)	Time (minutes)	Cycles
96	2	1
37	30	1

Table 11: Bead hybridization program

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

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

#尊敬的用户，由于网络监管政策的限制，部分内容暂时无法在本网站直接浏览。我们已经为您准备了相关原始数据和链接，感谢您的理解与支持。
<iframe title="YouTube video player" src="http://www.youtube.com/embed/_YQVbAMIqNM?si=d6zXFfntAC_9Xl_p" height="315" width="560"></iframe>