BHQplex™ CoPrimers™ enable multiplex PCR, whilst maintaining workflow flexibility. Their unique design allows assay interchangeability, overcoming a common shortfall of traditional multiplex PCR technologies. The added flexibility of interchangeability in turn supports a much broader range of applications including gene expression, adventitious presence (AP) testing, copy number variation, and SNP differentiation. Now you can think beyond the panel-based workflows for SNP genotyping, common in marker-assisted selection (MAS) breeding programs. Maximise multiplex PCR with BHQplex CoPrimers.
Bring flexibility to your lab with assay interchangeability
BHQplex CoPrimers address a critical shortfall of traditional multiplex PCR by enabling assay interchangeability. Traditional hydrolysis probe-based multiplex PCR requires extensive optimisation of assay pairs, and requires users to always run optimised pairs together. The highly selective nature of BHQplex CoPrimers enables users to interchange assays with one another, without the need for extensive re-optimisation of pairs. This maintains your laboratory’s flexibility, enabling a diverse set of projects with different marker sets to be run efficiently and cost effectively.
Assay interchangeability in bovine SNP genotyping
Interchangeability with BHQplex CoPrimers can be demonstrated with bovine DNA. Seven separate BHQplex CoPrimers assays were designed against purified bovine DNA, with one assay used in conjunction in a duplex SNP assay with each of the other four assays. Each assay was run in a single 5 µL reaction volume with BHQ® Probe Master Mix on a SNPline high-throughput instrument in 384-well plates in both SNP singleplex (2 targets) and SNP duplex (4 targets). Data was scored using Biosearch Technologies proprietary Laboratory Information Management System (LIMS), Kraken™, and compared to the original calls generated by KASP® genotyping. Examples of the end-point genotyping cluster plots is illustrated in Figure 2.
Figure 2: Purified bovine DNA (1.5 – 2.5 ng/µL) tested with BHQplex CoPrimers duplex assays labelled with (a) FAM™ and CAL Fluor® Orange 560 (assay rs29012457) and (b) TAMRA and Quasar 670 (assays rs29027559, DQ995976, ISAG-EF42090 and rs29001956). Blue = homozygous for FAM or TAMRA™ associated allele; red = homozygous for CAL Fluor Orange or Quasar® 670 associated allele; green = heterozygous; pink = unassigned genotypes; black = non-template controls.
In each cluster plot, genotypes can be confidently assigned, with little-to-no amplification of the non-template controls, showing limited cross-reactivity with assay interchangeability. In all instances, clear clusters representing the homozygous and heterozygous groups can be clearly identified, allowing for rapid genotype assignment for each sample tested.
As shown in Figure 3, the call rate for a singleplex assay was 99.8%. When this assay was then duplexed (Duplex 1, Duplex 2, Duplex 3 and Duplex 4), the call rate was >99.1%, with each assay subjected to a 4x4 assay interchangeable reaction. For all assays tested, for both BHQplex CoPrimers singleplex and BHQplex CoPrimers interchangeable duplex, 100% concordance in genotype assignment was observed when compared against KASP. Importantly, it should be noted that no optimisation was performed for any of the duplex reactions illustrated. These assays were designed using standard design algorithms, and run following the recommended annealing temperature of 55 °C, indicating that robust and consistent results can be obtained by interchanging the assays in each duplex reaction with no further optimisation required.
Figure 3: Purified bovine DNA was tested (n=94) with the background assay targeted with BHQplex CoPrimers labelled with FAM and CAL Fluor Orange 560. Call rate for the singleplex was determined by the mean of 19 assays. Each of the alternative assays in the duplex (rs29027559, DQ995976, EF042090 and rs29001956) were labelled with TAMRA and Quasar 670.
Interrogating multi-allelic SNPs common in agriculture species
One powerful application of BHQplex CoPrimers is the ease of detection of tetra-allelic SNPs. As seen in Figure 4 with purified maize DNA, the robustness of multiplexing combined with the flexibility demonstrated by the BHQplex CoPrimers interchangeability, allows for the combining of 2 assays (each targeting a bi-allelic SNP), allowing for the detection of 4 polymorphisms at single genomic target, within a single reaction. In all examples, 100% concordance was seen in genotype assignment when compared to KASP (call rate >97%).
Figure 4: Purified maize seed DNA tested with BHQplex CoPrimers assays, each targeted to the same genomic sequence. All reactions were run in 1.6 µL reaction volumes with BHQ Probe Master Mix on a Biosearch Technologies IntelliQube® instrument. Red = homozygous for FAM or TAMRA associated allele; blue = homozygous for CAL Fluor Orange or Quasar 670 associated allele; purple = heterozygous; black = non-template controls.
Real-time, Quantitative, and End-point PCR Applications
BHQplex CoPrimers is a versatile probe-like technology that is suitable for most PCR applications including Single Nucleotide Polymorphism (SNP) genotyping, Copy-Number Variation (CNV), and Adventitious Presence (AP) testing. Using the same PCR technology across all end-point and real-time PCR workflows in your lab provides the most-efficient operations. BHQplex CoPrimers can be designed for end-point and real-time PCR applications, and generate multiplex PCR savings throughout your lab.
Standard Curve comparisons with BHQplex CoPrimers in Singleplex and Multiplex qPCR
In 10 µL reaction volumes in 384-well plates on the BIO-RAD CFX the same bovine-specific BHQplex CoPrimers as used in Assay Interchangeability studies, was used to create both a standard curve, with final genomic DNA concentrations of 6 x 104 target copies to 6 x 100 target copies using a 4log10 range (which are typically used standard curve dilutions for qPCR applications) (Figure 5).
We selected a single bovine sample with a homozygous genotype for each target and created a single and multiplex standard curve using the same template input (Figure 5(a)). From this, confidence intervals between estimated and calculated values were generated (Figure 5(b)) together with measurements of the efficiency and linearity (Table 1). Using the standard curve, we performed absolute quantification using the same template at three different concentrations and determined estimated copies in both singleplex and multiplex. As with the previous reactions, 200 nM (final concentration) labelled BHQplex CoPrimers and 400 nM (final concentration) unlabelled Common Reverse BHQplex CoPrimers were used.
With standard curve generation, each assay was labelled with either FAM, CAL Fluor Red 610 or Quasar 670. Analysis of the data allowed for the calculation of both R2 and slope values (Figure 5(a)) and in all instances, R2>0.997, with a slope -3.289 to -3.315, indicating consistent and reproducible data for all dilutions tested. This highlights the sensitivity and specificity of the BHQplex CoPrimers assays, allowing for confidence in results generated even with as few as 6 target copies per reaction.
Figure 5(a): Standard curve generation for 3 bovine BHQplex CoPrimers multiplex assays (rs29027559, rs29012457 and DQ995976), in triplicate, each labelled with either FAM, CAL Fluor Red 610 and Quasar 670, respectively. For each 4log10 dilution-series a standard curve was generated, from which R2 and slope values were calculated.
For each of the assays targeted, the relative 95% confidence intervals were calculated via extrapolation from the generated standard curve with the percentage difference from reference vs qPCR estimation (Figure 5(b)). For all targets and for all copy numbers, there was little variation between the reference and the estimated values (no greater than 10% difference), showing accuracy of data generated, even at low copy number concentrations.
Figure 5(b): Confidence intervals (95%) for each of the assays, for a calculated and estimated copy number of 30, 300 and 3000 reference copy numbers per target (rs29027559, rs29012457 and DQ995976) with the percentage difference from reference vs qPCR estimation shown as %CV.
Calculations of efficiency and linearity for both singleplex and multiplex were determined (Table 1) from the standard curves generated in Figure 5. In either format, efficiency was shown to be between 90-110%, with all R2 values >0.997 down to an estimated 6 target copies. These highly reproducible data, even down to as low as 6 target copies per multiplex reaction, validate the use of BHQplex CoPrimers for the detection and quantification of low-copy number targets.
| Assay |
Dye |
Singleplex |
Multiplex |
Efficiency difference to multiplex (%) |
| Efficiency (%) |
Linearity (R2) |
Efficiency (%) |
Linearity (R2) |
| rs29027559 |
FAM |
97.6 |
0.999 |
101.4 |
0.999 |
3.8 |
| rs29012457 |
CAL Fluor Red |
106.3 |
0.998 |
101.1 |
0.998 |
>5.2 |
| DQ995976 |
Quasar 670 |
95.4 |
0.997 |
100.3 |
0.999 |
4.9 |
Table 1: Efficiency (%) and linearity (R2) calculations across the three bovine DNA targets (rs29027559, rs29012457 and DQ995976).
For research use only in Agrigenomic applications
