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Letters to the Editor |
Laboratory for Prenatal Medicine, University Women’s Hospital, Department of Research, Basel, Switzerland
aAddress correspondence to this author at: Dr. Sinuhe Hahn, Laboratory for Prenatal Medicine, University Women’s Hospital/Department of Research, Spitalstrasse 21, CH 4031 Basel, Switzerland. Fax 41-61-265-9399; e-mail shahn{at}uhbs.ch.
To the Editor:
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has recently emerged as a new platform for highly sensitive and accurate analysis of DNA, especially cell-free DNA (cf-DNA) (1)(2)(3). In this context MALDI-TOF MS has been shown to permit the detection of paternally inherited single nucleotide base variations from fetal cf-DNA in maternal plasma(2) and donor-derived single-base variations (also known as single-nucleotide polymorphisms) in the urine of kidney transplant recipients(3). Because these were small-scale studies, we conducted a larger study to determine the efficacy of this new methodology, particularly in comparison with the current standard, real-time PCR. Hence, we compared a MALDI-TOF MS assay with a well-established TaqMan® real-time PCR assay for the detection of the SRY (sex determining region Y) locus on the Y-chromosome(4).
After we obtained ethics approval from the Cantonal Institutional Review Board of Basel, we collected 97 maternal blood samples from healthy pregnant women (fetal gestational age 7–40 weeks: mean 17.6 weeks). The plasma was separated by centrifugation at 1600g for 10 min, then centrifuged again for 10 min at 16 000g (4). We used 500 µL of plasma for DNA isolation by use of the MagNa Pure DNA isolation instrument (Roche)(5). The cf-DNA was eluted into 100 µL elution buffer, of which 5 µL was used for each assay.
For the detection of the fetal SRY gene sequence by MALDI-TOF MS, we used a Sequenom MassArray® and an assay kindly provided by Sequenom Inc. Briefly, the SRY gene sequence was amplified with the following PCR primers: (forward) 5'-ACG TTG GAT GGT AAC GTT GAC TAC TTG CCC-3' and (reverse) 5'-ACG TTG GAT GCG CAT TTT TCA GGA CAG CAG-3'. The nonincorporated dNTPs from the amplification products were removed by shrimp alkaline phosphatase . Next, we carried out the homogenous MassEXTEND reaction containing the extension primer 5'-CAG GAC AGC AGT AGA GCA-3' and the terminator mix of ddGTP, dCTP, dTTP, and dATP. The extended products were cleaned up with SpectroCLEAN resin and dispensed onto a 384-format SpectroCHIP by a MassArray Nanodispenser. The spotted chip was read on a MassArray Analyzer Compact and analyzed with MassArray software (Sequenom. Inc). For the TaqMan real-time PCR analysis, the fetal SRY gene sequence was detected with a 7000 Sequence Detector (Perkin-Elmer Applied Biosystems) as previously described (4). All results of fetal sex determined by these 2 assays were confirmed by clinical data obtained postdelivery.
All the samples were examined in quadruplicate by the MALDI-TOF MS assay and the TaqMan real-time PCR assay in parallel. The presence or absence of the fetal SRY gene sequence was arbitrarily determined as follows: If the fetal SRY gene sequence was detectable in 2 or more of the 4 reactions, the result was scored as positive. Otherwise, it was considered to be negative. Our results indicated that the MALDI-TOF MS assay achieved a sensitivity of 95% (55/58), and the TaqMan real-time PCR assay yielded a sensitivity of 93% (54/58). The specificity of both assays was identical (Table 1
). The 3 false-negative cases examined by MALDI-TOF MS assay were also negative according to the TaqMan real-time PCR assay. The 3 samples were obtained from women during early pregnancy (gestational age: 11 weeks, 11.3 weeks, and 13.6 weeks, respectively). The other false-negative result occurring on the TaqMan real-time PCR assay was from a sample obtained at 17 weeks of gestation.
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In a comparative analysis, we observed that the MALDI-TOF MS assay and the TaqMan real-time PCR assay had similar detection rates, indicating that MALDI-TOF MS is as sensitive as TaqMan real-time PCR under these conditions. Hence, MALDI-TOF MS may be viewed as an attractive alternative to real-time PCR for the analysis of fetal cf-DNA in maternal plasma.
References
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