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 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.
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 (http://www.sigma-genosys.com/calc/DNACalc.asp) 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
2. Hybridization and Detection
3. Regeneration of Membrane
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.
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.
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.
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.
The authors have nothing to disclose.
Matthew O’Sullivan and Fei Zhou are recipients of Australian National Health and Medical Research Council Postgraduate Medical Research Scholarships.
Reagent | Company | Catalogue number | Comments |
---|---|---|---|
5′ amine C6 modified oligonucleotide probes | Sigma-Aldrich | ||
NaHCO3 | Sigma-Aldrich | S-8875 | 0.5 M solution made up to pH 8.4 |
NaOH | Sigma-Aldrich | S5881 | 0.1M solution |
20xSSPE buffer | Amresco | 0810-4L | |
Sodium dodecyl sulfate (SDS) | Sigma-Aldrich | L-4390 | Make up 10% stock solution, do not autoclave, should be kept for 1 week maximum before use |
Ethylenediaminetetraacetic acid (EDTA) | Sigma-Aldrich | E9884 | Make up 0.5 M solution adjusted to pH 8.0 and autoclave |
N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDAC) | Sigma-Aldrich | E7750 | Make up a 20 ml 16% solution just prior to use using 3.2 g EDAC and 18 ml Millipore water |
BiodyneC 0.45 μm nylon Membrane 20×20 cm | PALL life sciences | 74480C | |
Deionised, purified water | |||
Liquid Pyroneg detergent | Johnson Diversey | HH12291 | |
Streptavidin-peroxidase conjugate | Roche | 11 089 153 001 | |
Amersham ECL detection reagents | GE Healthcare | RPN2105 | |
Amersham ECL detection reagents | GE Healthcare | RPN2105 | |
OHP Transparency film | Corporate Express | EXP 504 OHP | |
Amersham hyperfilm ECL high performance chemiluminescent film 18×24 cm | GE Healthcare | 28906837 | |
Ice |
Table 1. Consumables used in the mPCR/RLB assay.
Equipment | Company | Catalogue number | Comments |
---|---|---|---|
Hybaid Shake’n’Stack Ovens (2) rolling bottle and nylon separating mesh | Thermo Scientific | HBSNSRS220 | Method can be performed with a single oven as long as there is facility for maintaining solutions at 42°C and 60°C prior to use |
The Belly Dancer rocking platform | Stovall life science | Euro BDbo | |
Miniblotter | Immunetics | MN100-45 | |
Water bath | |||
Suction | |||
Hot plate | |||
Exposure cartridge | Sigma-Aldrich | Z36,009-0 | |
X-ray film developer | 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 |
Table 2. Equipment required for the mPCR/RLB assay.