Simple and rapid detection of common fetal aneuploidies using peptide nucleic acid probe-based real-time polymerase chain reaction

Subeen Hong,Young Mi Jung,So Yeon Kim,Byoung Jae Kim,Sohee Oh,Chan-Wook Park,Jong Kwan Jun,Sun Min Kim,Joong Shin Park,Seung Mi Lee

doi:10.1038/s41598-021-02507-5

Abstract

To examine the detection performance of a peptide nucleic acid (PNA) probe-based real-time time polymerase chain reaction (PCR) assay to detect common aneuploidies. Using amniotic fluid samples, PNA probe based real-time PCR (Patio DEP Detection Kit; SeaSun Biomaterials, Korea) assay was performed. PNA probe was designed to hybridize to similar sequences located on different segments of target chromosomes (21, 18, and 13) and a reference chromosome. Amplification of target sequences and melting curve analysis was performed. When analyzing the melting curve, the ratio of the peak height of the target and reference chromosome was calculated and determined as aneuploidy if the ratio of peak height was abnormal. All the results from the PNA probe-based real-time PCR and melting curve analyses were compared to those from conventional karyotyping. Forty-two cases with common aneuploidies (24 of trisomy 21, 12 of trisomy 18, and 6 of trisomy 13) and 131 cases with normal karyotype were analyzed. When comparing the karyotyping results, the sensitivity and specificity of the PNA probe-based real-time PCR assay were both 100%. The level of agreement was almost perfect (k = 1.00). PNA real-time PCR assay is a rapid and easy method for detecting common aneuploidies.

Highlights

To examine the detection performance of a peptide nucleic acid (PNA) probe-based real-time time polymerase chain reaction (PCR) assay to detect common aneuploidies
A total of 173 amniotic fluid samples were used in the analysis, including 42 cases with common aneuploidies (24 with trisomy 21, 12 with trisomy 18, and 6 with trisomy 13) and 131 normal control cases
In Table 1. we summaries the detection performance of PNA probe-based real-time PCR compared with conventional karyotyping, when each trisomy was judged when the abnormal height ratio (K) was identified in three or more of the four sets

Summary

Introduction

To examine the detection performance of a peptide nucleic acid (PNA) probe-based real-time time polymerase chain reaction (PCR) assay to detect common aneuploidies. Karyotyping takes at least 2 weeks to yield results due to the requirement for culturing cell To overcome this limitation, rapid molecular methods such as fluorescence in situ hybridization (FISH), quantitative fluorescence polymerase chain reaction (QF-PCR) and multiplex ligation-dependent probe amplification (MLPA) have been d eveloped[1,2]. QF-PCR and MLPA are based on polymerase chain reaction (PCR) amplification of DNA short tandem repeat (STR) marker and of ligated probe, respectively[4,5] They require additional analysis after PCR using a genetic analyzer to determine sequence copy number. Peptide nucleic acid (PNA) probe-based real-time PCR, a simple and rapid detection test for fetal chromosomal abnormalities, was developed.

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Conclusion