Rapid Diagnosis of Aneuploidy Using Segmental Duplication Quantitative Fluorescent PCR

Abstract

The aim of this study was use a simple and rapid procedure, called segmental duplication quantitative fluorescent polymerase chain reaction (SD-QF-PCR), for the prenatal diagnosis of fetal chromosomal aneuploidies. This method is based on the co-amplification of segmental duplications located on two different chromosomes using a single pair of fluorescent primers. The PCR products of different sizes were subsequently analyzed through capillary electrophoresis, and the aneuploidies were determined based on the relative dosage between the two chromosomes. Each primer set, containing five pairs of primers, was designed to simultaneously detect aneuploidies located on chromosomes 21, 18, 13, X and Y in a single reaction. We applied these two primer sets to DNA samples isolated from individuals with trisomy 21 (n = 36); trisomy 18 (n = 6); trisomy 13 (n = 4); 45, X (n = 5); 47, XXX (n = 3); 48, XXYY (n = 2); and unaffected controls (n = 40). We evaluated the performance of this method using the karyotyping results. A correct and unambiguous diagnosis with 100% sensitivity and 100% specificity, was achieved for clinical samples examined. Thus, the present study demonstrates that SD-QF-PCR is a robust, rapid and sensitive method for the diagnosis of common aneuploidies, and these analyses can be performed in less than 4 hours for a single sample, providing a competitive alternative for routine use.

Segmental duplication-quantitative fluorescent-polymerase chain reaction: An approach for the diagnosis of Down syndrome in India

“…Genomic DNA isolation was performed using the standard phenol-chloroform method followed by PCR amplification using primers obtained from elsewhere [Muthuswamy and Agorwal ( 10 ) ]. The PCR conditions included initial denaturation at 95 °C for 5 minutes, followed by 35 cycles of 30 seconds at 95 °C, 30 seconds at 60 °C, and 30 seconds at 72 °C, and a final extension step at 72 °C for 10 minutes ( 9 , 10 ) . Amplified PCR products (2 µL) were denatured with 8 µL HiDI and 0.5 µL LIZ at 95 °C for 5 minutes and loaded onto the genetic analyzer (ABI 310 Genetic Analyzer, Applied Biosystems).…”

“…However, rapid aneuploidy testing methods such as fluorescent in situ hybridization (FISH), quantitative fluorescence-polymerase chain reaction (QF-PCR), and multiplex probe ligation assay (MLPA) are also routinely used for prenatal diagnosis in the laboratory ( 4 ) . A novel technique, segmental duplication-QF-PCR (SD-QF-PCR) was established by Kong et al ( 9 ) which involves SD sequences between test and control chromosomes to detect aneuploidies. SDs are two similar sequences with different fragment lengths, located on two different chromosomes.…”

“…The method involves amplifying SD regions. When these sequences are amplified using a single pair of fluorescent-labelled primers, the peak ratio between the two different chromosomes remains as 0.9 to 1.1, and trisomy 21 results in the ratio of 1.4 to 1.6 ( 9 , 10 ) .…”

Transfection with Livin and Survivin shRNA inhibits the growth and proliferation of non-small cell lung cancer cells

“…mRNA expression levels of Livin and Survivin. Fluorescent reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis was performed to determine the expression levels of Survivin and Livin according to a previous study (17). Following transfection for 72 h, the cells were harvested and total RNA extraction was performed using TRIzol reagent (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany).…”

Validação de teste de reação em cadeia da polimerase fluorescente quantitativa (QF-PCR) para detecção de aneuploidias fetais

“…Parte das alterações cromossômicas, as quais podem estar relacionadas a deficiências físicas e mentais, permanecerão não detectadas mesmo pela técnica padrão ouro (6). Neste contexto, surge a necessidade da existência de métodos mais rápidos de diagnóstico em que não seja necessária a cultura de células, a fim de reduzir a ansiedade do casal (2,6,17,19,21,22,31,(40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50). Várias técnicas têm sido desenvolvidas para o diagnóstico rápido de aneuploidias em amostras pré-natais.…”