SPECIMEN-SPECIFIC qPCR MIXES FOR THE SENSITIVE, SPECIFIC DETECTION OF VIRAL SNPs & ANTIBIOTIC- RESISTANCE MARKERS FROM BLOOD AND PLASMA.
SPECIMEN-SPECIFIC qPCR MIXES FOR THE SENSITIVE, SPECIFIC DETECTION OF VIRAL SNPs & ANTIBIOTIC- RESISTANCE MARKERS FROM BLOOD AND PLASMA.
Evelyn Tait, Ph. D. ; Mihai-Nicolae Podaru, Ph. D. ; William Ferreira, Ph. D. ; Rosa Maria Porreca, Ph. D. ; Danny Filer, Michele Amasio, Ph. D.
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Background
SNP genotyping is routinely used to track and identify new variants of viral pathogens 1 . The monitoring of viral strains is important both for the tracking of virulence mutations within infectious viral populations, and for the identification of specific genotypes that represent a higher-risk to patients 2 . Drug resistance mutations (DRMs) in viral and bacterial populations are a public health threat for existing and emerging diseases and rapid identification of such mutations is increasingly important in the treatment of bacteraemia and sepsis 3 . Screening for these mutations allows for better clinical management and supports the development of next-generation drugs. These applications, especially in a point-of-care (POC) setting, require a rapid, simplified process that can reduce the time to result, ideally avoiding the need for extraction. However, inhibitors found in blood samples and carried through extraction can often compromise such assays, whilst sensitivity and specificity are also significant challenges. We have demonstrated that blood inhibitor tolerant Air-Dryable Direct DNA qPCR Blood (MDX092) and Lyo-Ready ™ Genotyping Direct qPCR Blood Mix (MDX128) are perfectly suited for highly sensitive and specific detection of antibiotic-resistance markers and viral SNPs. Together with dried-format compatibility and multiplexing capability, these mixes have the potential to greatly enhance throughput and decrease the time to result for clinical laboratories.
Sensitive Accurate, Reliable Results
Nucleic-acid Extraction
Standard qPCR
OR
Sample collection
Sensitive Accurate, Reliable Results
Direct qPCR
Compatibility with Direct and Indirect Workflows: High inhibitor tolerant formulations are compatible either with direct workflows or those that include extraction steps, making these chemistries suitable for point-of- care (POC) and high-throughput users.
1. Methods
4. Sensitivity
Air-drying (MDX092) MDX092 (5 µ l/Rxn) and 20x primer-probes (1 µ l/Rxn) were combined in a reaction mix and 6- µ l aliquots were dispensed into the relevant wells of a microtitre plate, which was incubated at 80 °C for 20 min in a convection oven. Air-dried reactions (that can be stored for up to 24 months at ambient temperature) were reconstituted using the relevant quantity of template in 20 µ l elution buffer and amplified using a Bio-Rad CFX96 C1000 Touch. Sample / Template Preparation
Using extracted gDNA, in wet AND dry format, sensitivity down to 1 cfu/Rxn was achieved for E. coli and S. aureus targets. Similarly, amplification from qPCR Extraction Control carrying Amp R showed 100% efficiency in wet and dry formats and detection down to 10 cfu/Rxn (Figure 4). Dry format allowed reactions to be reconstituted in 100% extracted sample, maximising sensitivity.
qPCR Amplification
Reconstitution in 20- µ l sample containing template
(A) E. coli 16-23S ITS
(B) S. aureus 16-23S ITS
(C) Amp R
Master mix air-dried with primers & probe and stored for up to 24 months at ambient temperature.
Where plasma or blood was used to test inhibitor-tolerance the donor sample was combined with qPCR Extraction Control (MDX026) and / or gDNA to mimic bacteraemia and deliver a known concentration of template in a final reaction volume of 20 µ l. qPCR The thermal profile used for each assay is given in the relevant figure legend. Typically an extended “hot start” of 10 min was used to ensure release of DNA from the extraction control and thorough denaturation of the template.
Wet Efficiency: 100.7% Dry Efficiency:104.3%
Wet Efficiency: 97.7% Dry Efficiency: 97.9%
Wet Efficiency: 100.9% Dry Efficiency:100.5%
2. Inhibitor Tolerance
Figure 3. Sensitivity & qPCR Efficiency - Wet vs Dry. Template conc. shown at cfu per 20- µ l reaction. Thermal profile: 10 min @ 95 °C followed by 45 cycles of 15 s at 95 °C & 60 s at 60 °C.
In order to be compatible with direct amplification from blood samples MDX092 had to be tolerant to at least 20% plasma. Therefore, qPCR Extraction Control (MDX026) carrying ampicillin resistance gene, Amp R , was combined with 20% plasma and S. aureus gDNA at 5 cfu/ µ l per reaction. Figure 1 shows the amplification of Amp R and S. aureus 16-23S ITS in 0% and 20% plasma.
5. Specificity
Lyo-Ready ™ Genotyping Direct qPCR Blood Mix (MDX128) formulation was optimised for specific amplification of single nucleotide polymorphisms (SNPs) also with high tolerance for blood inhibitors, making it highly suitable for discriminating between viral strains directly from blood samples. Here, MDX128 was used to specifically amplify A and C SNPs in Epstein Barr variants in the presence of 10% whole blood, while mixes from other suppliers failed to discriminate between these variants (Figure 5). In further testing MDX128 distinguished between these variants in 20% plasma.
Amp R
S. aureus ITS 16-23S
MDX128
Supplier K
No Inhibitor 20% Plasma
Such high levels of tolerance for inhibitors not only allowed for amplification directly from blood samples but would also support more robust amplification from purified samples with fewer false-negatives. Figure 1. Inhibitor Tolerance - 0% vs 20% Plasma in the Reaction. qPCR Extraction Control at ~ 5 cfu/ µ l was combined with 5 genome equivalents (ge) per µ l in a 20- µ l reaction. Thermal profile: 10 min @ 95 °C followed by 45 cycles of 15 s at 95 °C & 60 s at 60 °C.
Supplier T
Supplier Q
3. Multiplexing
To further test the utility of MDX092 Amp R and S. aureus 16-23S ITS assays were combined with an assay for E. coli 16-23S ITS in a triplex reaction. All 3 targets were detected at 5 cfu / µ l in 20% plasma (Reaction Conc.) with no significant change in Ct value (Figure 2)
All Variants - No Discrimination
Bacteraemia Triplex: 0% vs 20% Plasma
Figure 5. Inhibitor Tolerance - SNP discrimination in 10% Whole Blood. Blue: Variant 1 only VIC fluorescence (SNP C). Red: homozygous for FAM ® fluorescence (SNP A). Green: Variant 3 (SNP A & C). Black: NTCs or undetermined. 10% of whole human blood was tested with Lyo-Ready ™ Genotyping Direct qPCR Blood Mix (Top left) vs. other supplier mixes using Epstein Barr Virus synthetic variants. Thermal profile: Pre-read - 30 s @ 60 °C ; 10 min @ 95 °C followed by 50 cycles of 15 s at 95 °C & 30 s at 63.5 °C; Post-Read - 30 s @ 60 °C.
45.0
40.0
35.0
Epstein Barr Variant 1 - Assay C
Epstein Barr Variant 2 - Assay A
30.0
25.0
20.0
15.0
10.0
5.0 0.0
Amp R - Mx
S. aureus - Mx
E. coli - Mx
0% Plasma 20% Plasma
Figure 6. Inhibitor Tolerance - SNP discrimination in 20% Plasma . Blue: Variant 1 only VIC fluorescence (SNP C). Red: homozygous for FAM ® fluorescence (SNP A). Thermal profile: Pre-read - 30 s @ 60 °C ; 10 min @ 95 °C followed by 50 cycles of 15 s at 95 °C & 30 s at 63.5 °C; Post-Read - 30 s @ 60 °C..
Figure 2. Multiplexing & Inhibitor Tolerance - 0% vs 20% Plasma in the Reaction. qPCR Extraction Control and gDNA at template conc. shown per 20- µ l reaction. Thermal profile: 10 min @ 95 °C followed by 45 cycles of 15 s at 95 °C & 60 s at 60 °C. The error bars represent 2 standard deviations across 6 replicates for each target/sample combination.
Conclusions
References
1. Vogels, C. B. F. , Breban, M. I. , Ott, I. M. et al (2021) “Multiplex qPCR discriminates variants of concern to enhance global surveillance of SARS- CoV-2” PLoS Biology 19 , 1-12 2. Micalessi, I. M., Boulet, G. A. V., Bogers, J. J. et al (2011) “High-throughput detection, genotyping and quantification of the human papillomavirus using real-time PCR” Clinical Chemistry and Laboratory Medicine 50 , 655-661. 3. Waddington C, Carey M. E., Boinett, C. J. et al (2022) “Exploiting genomics to mitigate the public health impact of antimicrobial resistance.” Genome Medicine 14 , 15
• The high blood-specific inhibitor tolerance demonstrated here enabled qPCR applications either from extracted samples or directly from plasma and blood. • The optimised formulations showed high specificity and amplification efficiency in discriminating viral SNPs and the identification of bacterial species. • Compatible with dried formats, these qPCR mixes represent powerful tools for the development of rapid point-of-care testing direct from blood and plasma samples and broader high-throughput screening . • Highly sensitive detection of bacterial targets (< 1 cfu / µl) in wet and dry formats.
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