October 15, 2012 (Vol. 32, No. 18)

Gene-Analysis Studies Advanced by Use of Low-Volume High-Throughput Real-Time PCR

This article describes three gene-analysis studies using the Roche LightCycler® 1536 Real-Time PCR System (Figure 1). In the first featured study, French researchers used high-throughput real-time PCR to test for the bacterial subtypes responsible for hemolytic-uremic syndrome (HUS)-causing enterohemorrhagic E. coli (Serovar EHEC O26:H11) pathogen, by genotyping nucleotide variations in the arcA gene.

In the second study, another French group, this time oncologists at the CRICM in Paris, reported using a new PCR assay with the LightCycler 1536 System to quantify rare blood circulating tumor DNA mutations. Using this new test, rare IDH1 mutations could be detected against a high background of DNA, they reported.

Finally, in a third study, researchers coupled the LightCycler 1536 High Throughput Real-Time PCR System with the Echo 555 Liquid Handler (Labcyte) in a workflow designed to demonstrate precision and reproducibility of system with low-volume samples.

Figure 1. The LightCycler® 1536 Instrument

Discriminating EHEC & EPEC Subtypes

Infections with E. coli, 026:H11 can cause severe diarrhea and hemolytic-uremic syndrome. Infection severity has been shown to be dependent on pathogen subtype. While EPEC subtypes produce diarrhea in children, the even more dangerous EHEC subtypes can produce bloody inflammation of the bowel, thrombocytopenia, and kidney dysfunction.

EHEC pathogens produced 56 fatalities in an outbreak in Germany in the summer of 2011. Using traditional microbiological and biochemical tests, it is not possible to differentiate EHEC and EPEC subtypes. Genotyping the pathogenic E. coli genes wzxO26, flic, eae-beta, stx, espK, and arcA has been established as the most reliable way to identify most subtypes.

While wzxO26, flic, eae-beta, stx, and espK can be identified using high-throughput qPCR, it was not previously possible to use a routine qPCR method to differentiate EHEC and EPEC subtypes based on the single base variation (SNP) in the E. coli arcA gene. Researchers at the Agence Nationale de Sécurité Sanitaire de l’Alimentation de l’Environnement et du Travail (ANSES), headed by Sabine Delannoy, have recently shown that arcA genotyping using the LightCycler 1536 Real Time PCR System is fast, accurate, and reproducible.

Using this system, 148 E. coli subtypes were tested in duplicate. Each haplotype used had previously been sequenced. At the same time, 33 O26:H11 subtypes of unknown genotype were also characterized. For real-time PCR, the Bravo Liquid Dispensing platform (Agilent) was used to dispense 1 µL volumes per well. The PCR test used primers specifically designed to allow target detection by a MGB (minor groove binder)-conjugated probe, or alternatively, by hydrolysis probes based on locked nucleic acid (LNA) technology.

Altogether, 769 samples were tested in duplicate, with no PCR failures. Detection sensitivity was very high. The 0.1 to 1.0 ng DNA templates used generated crossing point (Cp) values of 19.2 to 27.4 using MGB probes, and 18.9 to 26.3 using LNA probes. Delannoy et al. reported the high accuracy obtained in their results. A 100% correspondence was found for the genotyping results of the 148 samples when comparing results produced by the LightCycler 1536 System and DNA sequencing. Requiring just 90 min, the test was shown to be suitable for rapid high-throughput screening.

Genotyping Cancer Biomarkers Using COLD PCR and Digital PCR

Gene mutations for the enzyme isocitrate dehydrogenase 1 (IDH1) are reported to be associated with a positive survival prognosis in glioma patients. Patients with brain tumor cells carrying an IDH1 point mutation have been shown to exhibit a higher life expectancy than individuals with the wild type gene. Conventional PCR followed by Sanger DNA sequencing cannot detect this rare IDH1 mutation against a 20% background of wild type DNA. To overcome this problem, Boisellier et al. at the CRICM in Paris used two-step PCR to enrich IDH1 mutations in tumor DNA circulating in blood plasma.

Their sensitive genotyping assay required reliable detection of the single nucleotide difference between the IDH1 wild type and mutant using melting curve analysis (Figure 2). To detect the biomarker in blood samples, Boissellier et al. first extracted DNA from circulating blood, and then used their two-step PCR procedure. The extracted DNA was then selectively enriched for the rare IDH1 mutation using COLD-PCR.

Detected IDH1 mutations were quantified using clonal amplification, directly on the LightCycler 1536 Well Plates of the LightCycler 1536 Real-Time PCR System. Previously selectively amplified mutant IDH1 DNA was diluted 1 to 100,000, so that statistically only 0.5 to 1 DNA molecule was to be expected in each well. This dilution made a Yes/No quantification (0=absent, 1=present) of the IDH1 mutation possible using digital PCR. These researchers report that this procedure is not restricted to detection of the IDH1 biomarker and can be used in other instances requiring biomarker enrichment and quantification.

Figure 2. Representative heat maps obtained in an IDH1 normal subject (A) and an IDH1 mutant subject (B). Amplifications with positive and negative wells appear in green and in red, respectively.

Automated Low Volume High-Throughput qPCR Workflow

PCR volume miniaturization using the LightCycler 1536 Real-Time PCR System significantly conserves sample material, probes, and reaction components used, and also reduces setup and run time. Coupling the system to an automated liquid-handling device, such as the Echo 555 (Labcyte) enabling contact-free tipless dispensing, results in substantial additional cost and time savings, at the same time reducing the risk of cross-contamination.

Systematic testing has shown that the Labcyte system functions with a pipetting coefficient of variation of 0.011 µL for a final volume of 0.5 µL, and 0.025 µL for 1 µL final volume. Combining the Echo 555 and LightCycler 1536 Systems thus produces a highly reproducible PCR workflow for genotyping and gene expression analysis.

Thomas Froehlich, Ph.D. ([email protected]), is director R&D qPCR systems at Roche Applied Science.

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