PCR amplification of a triple-repeat genetic target directly from whole blood in 15 minutes as a proof-of-principle PCR study for direct sample analysis for a clinically relevant target

Abstract

BackgroundMost PCR-based diagnostics are still considered time- and labor-intensive due to disparate purification, amplification, and detection steps. Advancements in PCR enzymes and buffer chemistry have increased inhibitor tolerance, facilitating PCR directly from crude samples. Obviating the need for DNA purification, while lacking a concentration step, these direct sample methods are particularly apt for human genetic testing. However, direct PCR protocols have traditionally employed thermal cyclers with slow ramp rates and conservative hold times that significantly increase an assay’s time-to-result. For this proof-of-principle study, our objective was to significantly reduce sample preparation and assay time for a PCR-based genetic test, for myotonic dystrophy type 1 (DM1), by pairing an inhibitor-resistant enzyme mix with a rapid thermal cycler to analyze samples directly in whole blood.MethodsDM1 genetic screening was done with an adapted conventional PCR approach that employed the Streck Philisa® Thermal Cycler, the inhibitor-resistant NEBNext® High-Fidelity 2X PCR Master Mix, and agarose gel electrophoresis or an Agilent 2100 Bioanalyzer for detection. The Gene Link™ Myotonic Dystrophy Genemer™ Kit was used as a reference assay kit to evaluate the rapid assay.ResultsIn this work, a rapid and direct PCR assay testing 10% whole blood as template has been developed as an exclusionary screening assay for DM1, a triple-repeat genetic disorder. PCR amplification was completed in 15 minutes using 30 cycles, including in situ hot-start/cell lysis. Out of the 40 donors screened, this assay identified 23 (57.5%) as DM1 negative suggesting no need for further testing. These data are 100% concordant with data collected using the commercially available Gene Link Genemer™ Kit per the kit-specific PCR protocol.ConclusionsThe PCR assay described in this study amplified DM1 short tandem repeats in 15 minutes. By eliminating sample purification and slower conventional PCR protocols, we demonstrated how adaptation of current PCR technology and chemistries can produce a simple-to-use exclusionary screening assay that is independent of up-front sample prep, improving a clinical lab technician’s time-to-result. We envision this direct and rapid methodology could be applied to other conventional PCR-based genetic tests and sample matrices where genomic DNA is targeted for analysis within a given molecular diagnostic platform.

Inhibition mechanisms of hemoglobin, immunoglobulin G, and whole blood in digital and real-time PCR

“…Direct PCR analysis of blood samples is very appealing since it would reduce the cost and the time to results. The work done to improve the blood tolerance in PCR to allow direct analysis has included finding alternative DNA polymerases, engineering DNA polymerases, and applying various facilitators [ 1 , 2 , 11 – 13 , 43 , 45 , 47 ]. We have contributed to this field by elucidating the inhibitor mechanisms of blood and blood components in dPCR and qPCR.…”

“…Blood samples are widely used for polymerase chain reaction (PCR) analysis in fields such as diagnosis of infectious and genetic diseases in clinical medicine and forensic genetics. Direct PCR analysis with blood, without prior DNA extraction and purification, has been attempted to save time and reduce costs in routine analysis [ 1 – 6 ]. This approach is promising but is still limited by PCR inhibition induced by blood compounds [ 7 , 8 ].…”

Myotonic dystrophy: disease repeat range, penetrance, age of onset, and relationship between repeat size and phenotypes

“…Finally, an approach combining rolling circle amplification and Southern blotting to identify expanded repeats in DM1 and DM2 with low DNA input requirements [53] and a method which utilizes a special PCR enzyme mix and machine to screen normal DMPK alleles in less than 15 minutes have been described [•54]. …”

A Novel PCR Method for Detecting ACE Gene Insertion/Deletion Polymorphisms and its Clinical Application

“…Second, by using NaOH-treated blood samples for PCR, the problem of cross-contamination, which exists in traditional blood DNA purification processes is eliminated. Furthermore, various PCR inhibitors present in the samples such as hemoglobin, IgG, lactoferrin, and proteases are inactivated by NaOH treatment [ 29 , 30 ]. When the direct PCR product is loaded onto the sample pad of the LFA strip, certain impurities such as hemoglobin in the blood are filtered by the sample pad and conjugate pad, hence the chromatography is not affected.…”