TY - JOUR

T1 - Removal of Polymerase Chain Reaction Inhibitors by Electromembrane Extraction

AU - Liu, Cong

AU - Huang, Daixin

AU - Yang, Liuqian

AU - Wu, Shifan

AU - Shen, Xiantao

AU - Pedersen-Bjergaard, Stig

AU - Huang, Chuixiu

N1 - Publisher Copyright: ©

PY - 2021

Y1 - 2021

N2 - Polymerase chain reaction (PCR) technology has become the cornerstone of DNA analysis. However, special samples (e.g., forensic samples, soil, food, and mineral medicine) may contain powerful PCR inhibitors. High levels of inhibitors can hardly be sufficiently removed by conventional DNA extraction approaches and may result in the complete failure of PCR. In this work, the removal of PCR inhibitors by electromembrane extraction (EME) was investigated for the first time. To demonstrate the universality of the approach, EME formats with and without supported membranes (termed parallel-EME and μ-EME, respectively) were employed, and both anionic [humic acid (HA)] and cationic (Ca2+) PCR inhibitors were used as models. During EME, charged inhibitors in the sample migrate into the liquid membrane in the presence of an electric field and might further leech into the waste solution, while PCR analytes remain in the sample. After EME, the clearance values for HA at 0.2 and 2.5 mg mL-1 were 94 and 85%, respectively, and that for Ca2+ (275 mM) was 63%. Forensic PCR-short tandem repeat (PCR-STR) genotyping showed that EME significantly reduced the interference by HA in PCR-STR analysis and displayed a higher HA purge capability compared to existing methods. Furthermore, by combining EME with liquid-liquid extraction or solid-phase extraction, satisfactory STR profiles were obtained from HA-rich blood samples. In addition, false-negative reports of bacterial detection in mineral medicine and shrimps were avoided after the removal of Ca2+ by μ-EME. Our research demonstrates the great potential of EME for the purification of DNA samples containing high-level PCR inhibitors and opens up a new application direction for EME.

AB - Polymerase chain reaction (PCR) technology has become the cornerstone of DNA analysis. However, special samples (e.g., forensic samples, soil, food, and mineral medicine) may contain powerful PCR inhibitors. High levels of inhibitors can hardly be sufficiently removed by conventional DNA extraction approaches and may result in the complete failure of PCR. In this work, the removal of PCR inhibitors by electromembrane extraction (EME) was investigated for the first time. To demonstrate the universality of the approach, EME formats with and without supported membranes (termed parallel-EME and μ-EME, respectively) were employed, and both anionic [humic acid (HA)] and cationic (Ca2+) PCR inhibitors were used as models. During EME, charged inhibitors in the sample migrate into the liquid membrane in the presence of an electric field and might further leech into the waste solution, while PCR analytes remain in the sample. After EME, the clearance values for HA at 0.2 and 2.5 mg mL-1 were 94 and 85%, respectively, and that for Ca2+ (275 mM) was 63%. Forensic PCR-short tandem repeat (PCR-STR) genotyping showed that EME significantly reduced the interference by HA in PCR-STR analysis and displayed a higher HA purge capability compared to existing methods. Furthermore, by combining EME with liquid-liquid extraction or solid-phase extraction, satisfactory STR profiles were obtained from HA-rich blood samples. In addition, false-negative reports of bacterial detection in mineral medicine and shrimps were avoided after the removal of Ca2+ by μ-EME. Our research demonstrates the great potential of EME for the purification of DNA samples containing high-level PCR inhibitors and opens up a new application direction for EME.

U2 - 10.1021/acs.analchem.1c01689

DO - 10.1021/acs.analchem.1c01689

M3 - Journal article

C2 - 34383461

AN - SCOPUS:85114015701

VL - 93

SP - 11488

EP - 11496

JO - Industrial And Engineering Chemistry Analytical Edition

JF - Industrial And Engineering Chemistry Analytical Edition

SN - 0003-2700

IS - 33

ER -