Name: Multiplex PCR and Reverse Line Blot Hybridization Assay (mPCR/RLB) (Video) | JoVE Journal
Uploaded: 2011-08-06T13:00:00.000+00:00
Duration: 10 min 15 s
Description: Learn multiplex PCR and reverse line blot hybridization for detecting multiple pathogens from clinical samples. This cost-effective, high-throughput method enables simultaneous detection of up to 43 molecular targets without specialized equipment.

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5 minutes

Matthew O'Sullivan and Fei Zhou are recipients of Australian National Health and Medical Research Council Postgraduate Medical Research Scholarships.

Careful consideration must be given to primer and probe design. All available sequences of the targets of interest from databases such as GenBank should be utilized to identify conserved areas which are suitable targets. Where a large number of targets are being amplified in a single mPCR assay, each amplified sequence should be of similar length, and should not exceed 300 base pairs to avoid competition. An alternative application of the method is to amplify one longer target using primers against conserved regions and to use multiple probes to identify sequence variation within the amplicon. In this instance, the amplified PCR product may be longer. Primer annealing temperatures should all be similar, with PCR conditions adjusted accordingly. Primers which form strong secondary structure or primer dimer should be avoided. The Sigma Aldrich DNA calculator (<a href="http://www.sigma-genosys.com/calc/DNACalc.asp">http://www.sigma-genosys.com/calc/DNACalc.asp</a>) can be used to reliably predict these features.
DNA probes should be designed to have annealing temperatures close to 60°C. To maximize specificity, two probes for each target of interest can be included in the assay, one hybridizing to the forward DNA strand adjacent to the reverse primer binding site, and the other to the reverse strand adjacent to the forward primer binding site. In this case, both forward and reverse primers must be biotin-modified. If only one probe per amplified target is being used, then only one primer need be biotin modified. 
When designing primers and probes for the mPCR/RLB assay it is strongly advised to use in silico methods to predict PCR products and probe hybridization to correlate with the in vitro results. Ideally this is done using isolates for which the whole genome sequence is available. Software such as FastPCR (available at http://primerdigital.com/fastpcr.html) can be used for this purpose. Weak or absent probe signal can be predicted where in silico analysis indicates base pair mismatches resulting in low probe annealing temperatures.
DNA extraction techniques will vary depending on the samples being tested, and PCR conditions will be dependent on primer design. Readers are referred to publications on individual assays for more information regarding DNA extraction and PCR reagents and conditions1-19. 
Each assay is run with appropriate controls to provide at least one positive and one negative probe signal for each probe on the membrane, as well as a DNA-free control. Furthermore it is beneficial to include a probe at the top of the membrane that is expected to be positive for all samples (eg: a species or genus specific probe in the case of a micro-organism). This serves as a positive control probe, but also permits easy orientation of the results.
1.	Preparation of Membrane
<ol>
<li>Prepare the following solutions:
	<ul>
		<li>100ml 0.5M NaHCO3 (pH 8.4)</li>
 	<li>100 ml 0.5M NaHCO3</li>
		<li>20 ml 16% EDAC</li>
		<li>250 ml 0.1 M NaOH</li>
		<li>250 ml 2xSSPE</li>
		<li>250 ml 2xSSPE/0.1% SDS – place in waterbath at 60°C</li>
		<li>250 ml 20 mM EDTA.</li></ul></li>
<li>Pre-heat oven to 60°C.</li>
<li>Clean the Immunetics Miniblotter with 70% ethanol.</li>
<li>Dilute the oligonucleotide probes in 0.5 M NaHCO3 to a final concentration of 2 pmol/μl and a volume of 200 μl.</li>
<li>Cut a BiodyneC nylon membrane to 15x15 cm. Using a pencil and ruler, rule off a 0.5 cm space across top of membrane and write details here.</li>
<li>Seal membrane in plastic bag with 20 ml freshly made 16% EDAC solution and rock at room temperature for 10 minutes.</li>
<li>Wash membrane in deionised water for 30 seconds.</li>
<li>Place membrane in miniblotter with channels running across the membrane (parallel to pencil line). Put support cushion in place and close blotter. Aspirate fluid from channels.</li>
<li>Fill lanes 1 and 45 with 150 μl 0.5 M NaHCO3. Use 150 μl of each probe solution to fill lanes 2-44 in sequence, being careful to avoid air bubbles. If an air bubble does appear in the channel, keeping the pipette in place, rapidly aspirate the solution to allow the air bubble to float to the top of the pipette, then retry. Incubate at room temperature for 5 minutes.</li>
<li>Aspirate probe solutions, remove membrane and wash in 250 ml 0.1 M NaOH at room temperature for 9 minutes.</li>
<li>Wash membrane in 250 ml 2xSSPE for 30 seconds.</li>
<li>Wash membrane in 250 ml pre-warmed 2xSSPE/0.1% SDS in oven at 60°C for 5 minutes.</li>
<li>If not being used for hybridization immediately, wash membrane in 240 ml 20 mM EDTA at room temperature for 20 minutes (reserving remaining 10ml), then seal membrane in plastic bag with remaining 10 ml 20 mM EDTA and store in refrigerator at 4°C.</li>
<li>Wash miniblotter with Pyroneg detergent and brush. Rinse and allow to dry.</li>
</ol>
2.	 Hybridization and Detection
<ol>
<li>Prepare the following solutions:
	<ul>
		<li>250 ml 2xSSPE/0.1% SDS - store in water bath at 60°C.</li> 
		<li>500 ml 2xSSPE/0.5% SDS – store in water bath at 60°C</li>
		<li>500 ml 2xSSPE/0.5% SDS – store in oven at 42°C</li>
		<li>500 ml 2xSSPE - keep at room temperature</li>
		<li>500 ml 1% SDS – store in water bath at 60°C initially (for step 3)</li>
		<li>250 ml 20 mM EDTA – store at room temperature (for step 3).</li>
 </ul>
</li>
<li>Turn on one oven at 60°C and one oven to 42°C. Bring water to boil in a large beaker on a hotplate.</li>
<li>Clean Immunetics Miniblotter with 70% ethanol.</li>
<li>Aliquot 10 ml of 2xSSPE/0.1% SDS into a small container. Add 20 μl of each PCR product to 150 μl 2xSSPE/0.1% SDS in numbered tubes.</li> 
<li>Boil PCR products for 10 min at 100°C using styrofoam holders. Place on ice immediately for at least 5 minutes.</li>
<li>Wash membrane in remaining 240 ml 2xSSPE/0.1% SDS in 60°C oven for 5 minutes.</li>
<li>Place membrane in miniblotter with backing cushion. Orientate so channels run vertically (perpendicular to pencil line).</li>
<li>Suction excess liquid from miniblotter channels.</li>
<li>Add remaining 150 μl 2xSSPE/0.1% SDS to first and last channels. Add boiled PCR products to remaining channels (2-44) in sequence, being careful to avoid air bubbles.</li>
<li>Place miniblotter flat in 60°C oven for 1 hour to allow hybridization to take place.</li>
<li>Aspirate liquid from each channel then remove membrane from miniblotter.</li>
<li>Wash twice in prewarmed 250 ml 2xSSPE/0.5% SDS in 60°C oven for 10 minutes.</li>
<li>Moisten nylon separating mesh with 2xSSPE/0.5% SDS at 42°C and use it to roll up membrane.</li>
<li>Place rolled up membrane into roller tube, and unfurl to ensure membrane abuts the interior surface. Add 3 μl streptavidin-peroxidase conjugate (Roche Applied Science) to 15 ml 2xSSPE/0.5% SDS at 42°C and add to roller tube. Screw cap on tightly and incubate in roller oven at 42°C for 60 minutes. Ensure direction of roll is such that membrane does not tighten. Check periodically for leaks.</li>
<li>Wash miniblotter in Pyroneg detergent and water, rinse and allow to dry.</li>
<li>Remove membrane from roller tube. Wash twice in remaining 2xSSPE/0.5% SDS at 42°C for 10 minutes.</li>
<li>Wash twice in 2xSSPE at room temperature for 5 min.</li>
<li>Turn oven up to 80°C and place 500ml 1% SDS in oven to pre-warm.</li>
<li>Make up 15 ml Amersham ECL detection solution (7.5 ml of solution A and 7.5 ml of solution B). Discard 2xSSPE and add detection solution. Rock gently by hand for 2 minutes ensuring complete coverage of membrane with solution. Discard chemiluminescent solution.</li>
<li>Cut two sheets of plastic transparency to fit exposure cartridge. Place membrane between the two sheets and place into exposure cartridge.</li>
<li>Proceed to darkroom. Under red light, add ECL detection film to cartridge and expose for 5 minutes. Dog-ear lower right corner of film upon removal to allow correct orientation when viewing.</li>
<li>Develop film with automated developer or chemical baths as per manufacturer's directions.</li>
<li>Repeat exposure with longer or shorter exposure times if required.</li>
</ol>
3.	Regeneration of Membrane
<ol>
 <li>Wash membrane in 250 ml 1% SDS at 80°C for 30 minutes twice.</li>
 <li>Wash membrane in 240 ml 20 mM EDTA at room temperature for 15 minutes.</li>
 <li>Seal membrane in plastic bag with 10 ml 20 mM EDTA and refrigerate at 4°C for future re-use.</li>
</ol>
4. Representative Results: 
The results are best viewed by placing the film over a printed grid, or else scanning the image and importing into software such as BioNumerics (Applied Maths, Sint-Martens-Latem, Belgium). Each probe result should be interpreted with reference to positive and negative control probes. The results are best graded as negative, weak or positive. Positive results are where the signal is as strong as or stronger than the positive control probe. Negative results are where the signal is absent or equal to the negative control (in the case of background signal). Weak results are where the signal is fainter than the positive control probe, but stronger than the negative control. Weak results may be the result of point mutations leading to weak probe binding, or due to non-specific signals from primer dimer formation. Using two probes per target of interest can improve the specificity, where a single weak result can safely be interpreted as non-specific signal. If any doubt remains, a single-plex PCR reaction can be performed with gel-based detection and sequencing of any amplified product to determine if the result is truly positive. A representative result is shown in figure 2.
<img src="/files/ftp_upload/2781/2781fig1.jpg" alt="Amine-biotin-labeled DNA detection diagram; chemiluminescence on nylon membrane." data-altupdatedat="1788235205000" data-alt="Figure 1"> 
Figure 1. The mPCR/RLB principles (P1) Step 1.9. Amine modified probes are bound covalently to a nylon membrane. (P2) Step 2.10. Biotin modified PCR products are hybridized to the probes. (P3) Step 2.14. Streptavidin, labeled with peroxidase, is incubated with the membrane and binds to biotin. (P4) Steps 2.19-2.21. Peroxidase catalyses a reaction in the ECL detection reagents, producing light to which light sensitive film is exposed. The membrane is then washed for re-use. 
<img src="/files/ftp_upload/2781/2781fig2.jpg" alt="Dot blot analysis; grid of samples vs. probes; molecular interactions; diagram; data visualization." data-altupdatedat="1788235205000" data-alt="Figure 2"> 
Figure 2. Representative mPCR/RLB result. Samples 1 through 6 represent positive controls – between them there is at least one positive probe signal for each of the 43 probes. Each target sequence (A through U) is detected with two different probes to maximize specificity. Probes A1 and A2 represent species-specific probes which are expected to be positive for each sample and assist with orienting the film. Probe A1 is repeated at the bottom of the membrane. Samples 7 and 8 are negative controls. Note that Probes Q1, Q2, T1 and T2 have signal in the negative controls, indicating likely nonspecific binding of primers or primer dimer product. Re-design of these probes or primers would be required. Probes C1, D1 and F1 show streaking across the membrane likely due to some non-specific uptake of the streptavidin-peroxidase conjugate or chemiluminescent substrate, however this is easily distinguished from true positive probe signals. Probes D1 and D2 have relatively weak signal compared to other probes, this may be due to larger amplicons leading to less efficient amplification. Probe J2 is negative is several samples (1, 3, 4, 6, 39) where probe J1 is positive. This most likely represents a mutation in the J2 binding site for these samples.

<ol><li>O'Sullivan, M. V., Cai, Y., Kong, F., Zeng, X., Gilbert, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Influence+of+disk+separation+distance+on+accuracy+of+the+disk+approximation+test+for+detection+of+inducible+clindamycin+resistance+in+Staphylococcus+spp%5BTitle%5D)+AND+%22J+Clin+Microbiol%22%5BJournal%5D)">Influence of disk separation distance on accuracy of the disk approximation test for detection of inducible clindamycin resistance in Staphylococcus spp.</a> J Clin Microbiol. 44, 4072-4076 (2006).</li>
<li>Zeng, X., Kong, F., Wang, H., Darbar, A., Gilbert, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Simultaneous+detection+of+nine+antibiotic+resistance-related+genes+in+Streptococcus+agalactiae+using+multiplex+PCR+and+reverse+line+blot+hybridization+assay%5BTitle%5D)+AND+%22Antimicrob+Agents+Chemother%22%5BJournal%5D)">Simultaneous detection of nine antibiotic resistance-related genes in Streptococcus agalactiae using multiplex PCR and reverse line blot hybridization assay.</a> Antimicrob Agents Chemother. 50, 204-209 (2006).</li>
<li>Cai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+single-+and+multilocus+sequence+typing+and+toxin+gene+profiling+for+characterization+of+methicillin-resistant+Staphylococcus+aureus%5BTitle%5D)+AND+%22J+Clin+Microbiol%22%5BJournal%5D)">Comparison of single- and multilocus sequence typing and toxin gene profiling for characterization of methicillin-resistant Staphylococcus aureus.</a> J Clin Microbiol. 45, 3302-3308 (2007).</li>
<li>Cai, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+new+multiplex+PCR-based+reverse+line-blot+hybridization+%28mPCR%2FRLB%29+assay+for+rapid+staphylococcal+cassette+chromosome+mec+%28SCCmec%29+typing%5BTitle%5D)+AND+%22J+Med+Microbiol%22%5BJournal%5D)">A new multiplex PCR-based reverse line-blot hybridization (mPCR/RLB) assay for rapid staphylococcal cassette chromosome mec (SCCmec) typing.</a> J Med Microbiol. 58, 1045-1057 (2009).</li>
<li>O'Sullivan, M. V., Kong, F., Sintchenko, V., Gilbert, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+identification+of+methicillin-resistant+Staphylococcus+aureus+transmission+in+hospitals+by+use+of+phage-derived+open+reading+frame+typing+enhanced+by+multiplex+PCR+and+reverse+line+blot+assay%5BTitle%5D)+AND+%22J+Clin+Microbiol%22%5BJournal%5D)">Rapid identification of methicillin-resistant Staphylococcus aureus transmission in hospitals by use of phage-derived open reading frame typing enhanced by multiplex PCR and reverse line blot assay.</a> J Clin Microbiol. 48, 2741-2748 (2010).</li>
<li>Wang, Q., Kong, F., Jelfs, P., Gilbert, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extended+phage+locus+typing+of+Salmonella+enterica+serovar+Typhimurium%2C+using+multiplex+PCR-based+reverse+line+blot+hybridization%5BTitle%5D)+AND+%22J+Med+Microbiol%22%5BJournal%5D)">Extended phage locus typing of Salmonella enterica serovar Typhimurium, using multiplex PCR-based reverse line blot hybridization.</a> J Med Microbiol. 57, 827-838 (2008).</li>
<li>Zhou, F., Kong, F., Tong, Z., Gilbert, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+less-common+Streptococcus+pneumoniae+serotypes+by+a+multiplex+PCR-based+reverse+line+blot+hybridization+assay%5BTitle%5D)+AND+%22J+Clin+Microbiol%22%5BJournal%5D)">Identification of less-common Streptococcus pneumoniae serotypes by a multiplex PCR-based reverse line blot hybridization assay.</a> J Clin Microbiol. 45, 3411-3415 (2007).</li>
<li>Kong, F., Brown, M., Sabananthan, A., Zeng, X., Gilbert, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multiplex+PCR-based+reverse+line+blot+hybridization+assay+to+identify+23+Streptococcus+pneumoniae+polysaccharide+vaccine+serotypes%5BTitle%5D)+AND+%22J+Clin+Microbiol%22%5BJournal%5D)">Multiplex PCR-based reverse line blot hybridization assay to identify 23 Streptococcus pneumoniae polysaccharide vaccine serotypes.</a> J Clin Microbiol. 44, 1887-1891 (2006).</li>
<li>Kong, F., Ma, L., Gilbert, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Simultaneous+detection+and+serotype+identification+of+Streptococcus+agalactiae+using+multiplex+PCR+and+reverse+line+blot+hybridization%5BTitle%5D)+AND+%22J+Med+Microbiol%22%5BJournal%5D)">Simultaneous detection and serotype identification of Streptococcus agalactiae using multiplex PCR and reverse line blot hybridization.</a> J Med Microbiol. 54, 1133-1138 (2005).</li>
<li>Zhou, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+20+common+human+enterovirus+serotypes+by+use+of+a+reverse+transcription-PCR-based+reverse+line+blot+hybridization+assay%5BTitle%5D)+AND+%22J+Clin+Microbiol%22%5BJournal%5D)">Identification of 20 common human enterovirus serotypes by use of a reverse transcription-PCR-based reverse line blot hybridization assay.</a> J Clin Microbiol. 47, 2737-2743 (2009).</li>
<li>Zhou, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+identification+and+analysis+of+nonserotypeable+human+enteroviruses%5BTitle%5D)+AND+%22J+Clin+Microbiol%22%5BJournal%5D)">Molecular identification and analysis of nonserotypeable human enteroviruses.</a> J Clin Microbiol. 48, 1276-1282 (2010).</li>
<li>Xiong, L., Kong, F., Yang, Y., Cheng, J., Gilbert, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Use+of+PCR+and+reverse+line+blot+hybridization+macroarray+based+on+16S-23S+rRNA+gene+internal+transcribed+spacer+sequences+for+rapid+identification+of+34+mycobacterium+species%5BTitle%5D)+AND+%22J+Clin+Microbiol%22%5BJournal%5D)">Use of PCR and reverse line blot hybridization macroarray based on 16S-23S rRNA gene internal transcribed spacer sequences for rapid identification of 34 mycobacterium species.</a> J Clin Microbiol. 44, 3544-3550 (2006).</li>
<li>Wang, H., Kong, F., Wang, B., McKechnie, M. L., Gilbert, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multiplex+polymerase+chain+reaction-based+reverse+line+blot+hybridization+assay+to+detect+common+genital+pathogens%5BTitle%5D)+AND+%22Int+J+STD+AIDS%22%5BJournal%5D)">Multiplex polymerase chain reaction-based reverse line blot hybridization assay to detect common genital pathogens.</a> Int J STD AIDS. 21, 320-325 (2010).</li>
<li>McKechnie, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Simultaneous+identification+of+14+genital+microorganisms+in+urine+by+use+of+a+multiplex+PCR-based+reverse+line+blot+assay%5BTitle%5D)+AND+%22J+Clin+Microbiol%22%5BJournal%5D)">Simultaneous identification of 14 genital microorganisms in urine by use of a multiplex PCR-based reverse line blot assay.</a> J Clin Microbiol. 47, 1871-1877 (2009).</li>
<li>Xiong, L., Kong, F., Zhou, H., Gilbert, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Use+of+PCR+and+reverse+line+blot+hybridization+assay+for+rapid+simultaneous+detection+and+serovar+identification+of+Chlamydia+trachomatis%5BTitle%5D)+AND+%22J+Clin+Microbiol%22%5BJournal%5D)">Use of PCR and reverse line blot hybridization assay for rapid simultaneous detection and serovar identification of Chlamydia trachomatis.</a> J Clin Microbiol. 44, 1413-1418 (2006).</li>
<li>Wang, Y., Kong, F., Yang, Y., Gilbert, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+multiplex+PCR-based+reverse+line+blot+hybridization+%28mPCR%2FRLB%29+assay+for+detection+of+bacterial+respiratory+pathogens+in+children+with+pneumonia%5BTitle%5D)+AND+%22Pediatr+Pulmonol%22%5BJournal%5D)">A multiplex PCR-based reverse line blot hybridization (mPCR/RLB) assay for detection of bacterial respiratory pathogens in children with pneumonia.</a> Pediatr Pulmonol. 43, 150-159 (2008).</li>
<li>Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Use+of+a+multiplex+PCR-based+reverse+line+blot+%28mPCR%2FRLB%29+hybridisation+assay+for+the+rapid+identification+of+bacterial+pathogens%5BTitle%5D)+AND+%22Clin+Microbiol+Infect%22%5BJournal%5D)">Use of a multiplex PCR-based reverse line blot (mPCR/RLB) hybridisation assay for the rapid identification of bacterial pathogens.</a> Clin Microbiol Infect. 14, 155-160 (2008).</li>
<li>Wang, H., Kong, F., Jelfs, P., James, G., Gilbert, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Simultaneous+detection+and+identification+of+common+cell+culture+contaminant+and+pathogenic+mollicutes+strains+by+reverse+line+blot+hybridization%5BTitle%5D)+AND+%22Appl+Environ+Microbiol%22%5BJournal%5D)">Simultaneous detection and identification of common cell culture contaminant and pathogenic mollicutes strains by reverse line blot hybridization.</a> Appl Environ Microbiol. 70, 1483-1486 (2004).</li>
<li>Kong, F., Gilbert, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multiplex+PCR-based+reverse+line+blot+hybridization+assay+%28mPCR%2FRLB%29+-+a+practical+epidemiological+and+diagnostic+tool%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Multiplex PCR-based reverse line blot hybridization assay (mPCR/RLB) - a practical epidemiological and diagnostic tool.</a> Nat Protoc. 1, 2668-2680 (2006).</li>
</ol>

No conflicts of interest declared.

The mPCR/RLB method permits simultaneous detection of a large number of PCR amplicons. Because of the high sensitivity of chemiluminescent probe-based detection, a single multiplex PCR reaction with large numbers of primer pairs can be used to amplify the DNA template.
If no probe signals are obtained with one or more samples, performing gel electrophoresis with any remaining PCR product can help distinguish whether the problem is with PCR amplification or probe hybridization. Where all probe signals on the membrane are weak or absent, but gel electrophoresis indicates successful PCR amplification, possibilities include problems with labeling of the membrane, incorrect regeneration of the membrane after previous use, incorrect temperatures during hybridization or streptavidin incubation, or defective streptavidin-peroxidase conjugate or detection reagent.
If control samples indicate that individual probes may be producing false negative signals, single PCR with gel-based detection and subsequent sequencing can be performed to verify amplification of PCR product and to look for sequence variation at the probe binding site. Weak or absent probe signal may be due to large amplicon size: if possible all amplicons should be of similar length and less than 300 bp. Secondary DNA structures of amplicons may also produce poor probe binding, and may necessitate re-design of primers. Individual primer or probe concentrations may also be varied to optimize signal strength.
False positive signals due to probe binding of nonamplified primers or primer-dimer product can be investigated by performing single PCR with gel-based detection and subsequent sequencing. If this occurs, redesign of the probes may be necessitated. Point mutations in probe binding sites may lead to weak or absent signals. Allowing two probes for each amplified product makes this easy to detect. This characteristic may be exploited with careful primer and probe design to detect small sequence variations in target DNA.
In summary, while there are many methods of product detection following multiplex PCR reactions available, mPCR/RLB has the advantage of being high-throughput and inexpensive with low setup-costs. The flexibility of the method permits its use in for a wide range of applications.

<table><tbody><tr><td>5&amp;rsquo; amine C6 modified oligonucleotide probes</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S-8875</td><td>0.5 M solution made up to pH 8.4</td></tr><tr><td>NaOH</td><td>Sigma-Aldrich</td><td>S5881</td><td>0.1M solution</td></tr><tr><td>20xSSPE buffer</td><td>Amresco</td><td>0810-4L</td><td></td></tr><tr><td>Sodium dodecyl sulfate (SDS)</td><td>Sigma-Aldrich</td><td>L-4390</td><td>Make up 10% stock solution, do not autoclave, should be kept for 1 week maximum before use</td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma-Aldrich</td><td>E9884</td><td>Make up 0.5 M solution adjusted to pH 8.0 and autoclave</td></tr><tr><td>N-(3-Dimethylaminopropyl)-N&amp;#8242;-ethylcarbodiimide hydrochloride (EDAC)</td><td>Sigma-Aldrich</td><td>E7750</td><td>Make up a 20 ml 16% solution just prior to use using 3.2 g EDAC and 18 ml Millipore water</td></tr><tr><td>BiodyneC 0.45 &amp;mu;m nylon Membrane 20x20 cm</td><td>Pall Corporation</td><td>74480C</td><td></td></tr><tr><td>Deionised, purified water</td><td></td><td></td><td></td></tr><tr><td>Liquid Pyroneg detergent</td><td>Johnson Diversey</td><td>HH12291</td><td></td></tr><tr><td>Streptavidin-peroxidase conjugate</td><td>Roche Group</td><td>11 089 153 001</td><td></td></tr><tr><td>Amersham ECL detection reagents</td><td>GE Healthcare</td><td>RPN2105</td><td></td></tr><tr><td>Amersham ECL detection reagents</td><td>GE Healthcare</td><td>RPN2105</td><td></td></tr><tr><td>OHP Transparency film</td><td>Corporate Express</td><td>EXP 504 OHP</td><td></td></tr><tr><td>Amersham hyperfilm ECL high performance chemiluminescent film 18x24 cm</td><td>GE Healthcare</td><td>28906837</td><td></td></tr><tr><td>Ice</td><td></td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Table 1.&lt;/strong&gt; Consumables used in the mPCR/RLB assay.</td></tr><tr><td>Hybaid Shake&amp;rsquo;n&amp;rsquo;Stack Ovens (2) rolling bottle and nylon separating mesh</td><td>Thermo Fisher Scientific, Inc.</td><td>HBSNSRS220</td><td>Method can be performed with a single oven as long as there is facility for maintaining solutions at 42&amp;#x00B0;C and 60&amp;#x00B0;C prior to use</td></tr><tr><td>The Belly Dancer rocking platform</td><td>Stovall Life Sciences, IBI Sciences</td><td>Euro BDbo</td><td></td></tr><tr><td>Miniblotter</td><td>Immunetics</td><td>MN100-45</td><td></td></tr><tr><td>Water bath</td><td></td><td></td><td></td></tr><tr><td>Suction</td><td></td><td></td><td></td></tr><tr><td>Hot plate</td><td></td><td></td><td></td></tr><tr><td>Exposure cartridge</td><td>Sigma-Aldrich</td><td>Z36,009-0</td><td></td></tr><tr><td>X-ray film developer</td><td></td><td></td><td>Can also use developer, fixer and water in trays in dark room instead of automated developer. Lumino-imager may also be used instead of Xray film</td></tr><tr><td colspan="4">&lt;strong&gt;Table 2.&lt;/strong&gt; Equipment required for the mPCR/RLB assay.</td></tr></tbody></table>

multiplex pcr and reverse line blot hybridization assay (mpcr/rlb)

Multiplex PCR and Reverse Line Blot Hybridization Assay (mPCR/RLB)

Multiplex PCR/Reverse Line Blot Hybridization assay allows the detection of up to 43 molecular targets in 43 samples using one multiplex PCR reaction followed by probe hybridization on a nylon membrane, which is re-usable. Probes are 5' amine modified to allow fixation to the membrane. Primers are 5' biotin modified which allows detection of hybridized PCR products using streptavidin-peroxidase and a chemiluminescent substrate via photosensitive film. With low setup and consumable costs, this technique is inexpensive (approximately US$2 per sample), high throughput (multiple membranes can be processed simultaneously) and has a short turnaround time (approximately 10 hours). 
The technique can be utilized in a number of ways. Multiple probes can be designed to detect sequence variation within a single amplified product, or multiple products can be amplified
simultaneously, with one (or more) probes used for subsequent detection. A combination of both approaches can also be used within a single assay. The ability to include multiple probes for a single target sequence makes the assay highly specific.
Published applications of mPCR/RLB include detection of antibiotic resistance genes1,2, typing of methicillin-resistant Staphylococcus aureus3-5 and Salmonella sp6, molecular serotyping of Streptococcus pneumoniae7,8, Streptococcus agalactiae9 and enteroviruses10,11, identification of Mycobacterium sp12, detection of genital13-15 and respiratory tract16 and other17 pathogens and detection and identification of mollicutes18. However, the versatility of the technique means the applications are virtually limitless and not restricted to molecular analysis of micro-organisms.
The five steps in mPCR/RLB are a) Primer and Probe design, b) DNA extraction and PCR amplification c) Preparation of the membrane, d) Hybridization and detection, and e) Regeneration of the Membrane.

An inexpensive, high throughput method for simultaneous detection of up to 43 molecular targets is described. Applications of mPCR/RLB include microbial typing and detection of multiple pathogens from clinical samples.

Multiplex PCR/Reverse Line Blot Hybridization assay allows the detection of up to 43 molecular targets in 43 samples using one multiplex PCR reaction ...

The overall goal of this procedure is to determine the presence or absence of a panel of specific nucleic acid sequences previously amplified in a multiplex PCR reaction. This is accomplished by first fixing amine labeled DNA probes to a reusable nylon membrane. Biotin labeled PCR products are then hybridized to the membrane bound probes and strep TTR in peroxidase.Conjugate is added to the bound PCR products. Next, the bound PCR products are detected by adding chemiluminescence detection reagents, and exposing the membrane to high sensitivity x-ray film. Finally, the membrane is washed to remove the hybridized PCR products, allowing it to be reused for future assays.Ultimately, results can be obtained that show hybridization of PCR products to the membrane membrane-bound probes on the x-ray film. The main advantages of this technique over existing methods such as multiplex PCR, with gel electrophoresis real-time PCR or microarray, is that it's high throughput, relatively rapid, does not require any specialized equipment, and most importantly is inexpensive. Before beginning, prepare the following solutions according to the written portion of this protocol.To prepare the membrane, preheat the oven to 60 degrees Celsius. Clean the mini blotter with 70%ethanol, dilute the oligonucleotide probes in 0.5 molar sodium bicarbonate to a final concentration of two picomoles per microliter and a volume of 200 microliters. Cut the nylon membrane to 15 by 15 centimeters using a pencil and ruler mark off a 0.5 centimeter space across the top of the membrane, and write the details of the experiment there.Seal the membrane in a plastic bag with 20 milliliters of freshly made 16%EDAC solution and rock it room temperature for 10 minutes. Wash the membrane in deionized water for 30 seconds. Next, place the membrane in a mini blotter with channels running across the membrane.Then put the support cushion in place and close the blotter. Thoroughly aspirate any fluid from the channels. Fill lanes one and 45 with 150 microliters of 0.5 molar sodium bicarbonate.Use 150 microliters of each probe solution to fill lanes two to 44 in sequence being careful to avoid air bubbles. If an air bubble does appear in the channel, keeping the pipette in place rapidly aspirate the solution to allow the air bubble to float to the top of the pipette tip, then retry. Incubate at room temperature for five minutes.Aspirate the probe solutions. Remove the membrane and wash it in 250 milliliters of 0.1 molar sodium hydroxide at room temperature for nine minutes. Wash the membrane in 250 milliliters of two times SSPE for 30 seconds.Then wash it in 250 milliliters of prewarm two times SSPE 0.1%SDS in the oven at 60 degrees Celsius for five minutes, if not immediately being used for hybridization. Wash the membrane in 240 milliliters of 20 millimolar EDTA at room temperature for 20 minutes. Then sell the membrane in a plastic bag with the remaining 10 milliliters of 20 millimolar EDTA and store at four degrees Celsius.Finally, wash the mini blosser with pyro neck detergent and a brush rinse and allow it to dry multiplex. PCR Amplification of the targets of interest is not described in this demonstration, but must be performed prior to pro hybridization. Before beginning the hybridization procedure, prepare the following solutions according to the written portion of this protocol.Turn on one oven and set to 60 degrees Celsius and another oven to 42 degrees Celsius. Bring the water to a boil in a large beaker on a hot plate. Clean the mini blotter with 70%ethanol.Next aliquot 10 milliliters of two times SSPE 0.1%SDS into a small container. Then add 20 microliters of each PCR product to 150 microliters of two times SSPE 0.1%SDS in numbered tubes. Boil the PCR products for 10 minutes at 100 degrees Celsius using styrofoam holders place on ice immediately for at least five minutes.Wash the membrane in the remaining 240 milliliters of two times SSPE 0.1%SDS in the 60 degree Celsius oven for five minutes. Place the membrane in the mini blotter with backing cushion. Orient it so that the channels run vertically.Suction the excess liquid from the mini blotter channels. Add the remaining 150 microliters of two times SSPE 0.1%SDS to the first and last channels. Add boiled PCR products to channels two to 44 in sequence being careful to avoid air bubbles.Place the mini blotter flat in the 60 degree celsius oven for one hour to allow hybridization to take place. Spray the liquid from each channel. Then remove the membrane from the mini blotter.Washed twice in pre-warned 250 milliliters of two times SSPE, 0.5%SDS in the 60 degree Celsius oven for 10 minutes. Moisten the nylon backing with two times SSPE 0.5%SDS at 42 degrees Celsius and use it to roll up the membrane. Place the rolled up membrane into a roller tube and unfer to ensure the membrane abuts the interior surface.Add three microliters of strept AD in peroxidase conjugate to 15 milliliters of two times SSPE, 0.5%SDS at 42 degrees Celsius and add to the tube. Screw the cap on tightly and incubate in the roller oven at 42 degrees Celsius for 60 minutes. Ensure the direction of the roll is such that the membrane does not tighten.Check periodically for leaks. Wash the mini blotter in pyro neck detergent and water rinse and allow it to dry. Remove the membrane from the roller tube wash twice in the remaining two times SSPE 0.5%SDS at 42 degrees Celsius for 10 minutes.Then wash twice in two times SSPE at room temperature for five minutes. Turn both ovens up to 80 degrees Celsius and place 500 milliliters of 1%SDS in one oven to prewarm. Make up the ECL solution and incubate the membrane in ECL detection solution for two minutes.Then expose the membrane to film and develop. Repeat the exposure with longer or shorter times if required to regenerate the membrane. Wash it in 250 milliliters of 1%SDS at 80 degrees Celsius for 30 minutes twice.Then Washington 240 milliliters of 20 millimolar EDTA at room temperature for 15 minutes. Seal the membrane in a plastic bag with 10 milliliters of 20 millimolar EDTA and refrigerator at four degrees Celsius for future reuse. This figure shows the results from a multiplex PCR reverse line blot hybridization assay Samples one through six represent positive controls between them.There is at least one positive probe signal for each of the 43 probes. Each target sequence A through U is detected with two different probes to maximize specificity probes. A one and a two represents species specific probes, which are expected to be positive for each sample and assist with orienting the film probe.A one is repeated at the bottom of the membrane. Samples seven and eight are negative controls. Note that probes Q1, Q2, T one and T two have signal in the negative controls indicating likely nonspecific binding of primers or primer dimer.Product redesign of these probes or primers would be required probes, C one, D one and F1 show streaking across the membrane, likely due to some non-specific uptake of the strep TD in peroxidase, conjugate, or chemiluminescent substrate. However, this is easily distinguished from true positive probes. Signals probes D one and D two have relatively weak signals compared to the other probes.This may be due to larger amplicons leading to less efficient amplification. Finally, probe J two is negative in several samples where probe J one is positive. This most likely represents a mutation in the J two binding site for these samples.Once mastered this technique from DNA extraction through PCR Pro hybridization, result interpretation and regeneration of the membrane can all be done in less than 10 hours.

multiplex-pcr-and-reverse-line-blot-hybridization-assay-mpcrrlb

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Learn multiplex PCR and reverse line blot hybridization for detecting multiple pathogens from clinical samples. This cost-effective, high-throughput method enables simultaneous detection of up to 43 molecular targets without specialized equipment.

Multiplex PCR and Reverse Line Blot Hybridization Assay (mPCR/RLB)

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