Accurate Quantification of Microorganisms in PCR-Inhibiting Environmental DNA Extracts by a Novel Internal Amplification Control Approach Using Biotrove OpenArrays

Abstract

PCR-based detection assays are prone to inhibition by substances present in environmental samples, thereby potentially leading to inaccurate target quantification or false-negative results. Internal amplification controls (IACs) have been developed to help alleviate this problem but are generally applied in a single concentration, thereby yielding less-than-optimal results across the wide range of microbial gene target concentrations possible in environmental samples (J. Hoorfar, B. Malorny, A. Abdulmawjood, N. Cook, M. Wagner, and P. Fach, J. Clin. Microbiol. 42:1863-1868, 2004). Increasing the number of IACs for each quantitative PCR (qPCR) sample individually, however, typically reduces sensitivity and, more importantly, the reliability of quantification. Fortunately, current advances in high-throughput qPCR platforms offer the possibility of multiple reactions for a single sample simultaneously, thereby allowing the implementation of more than one IAC concentration per sample. Here, we describe the development of a novel IAC approach that is specifically designed for the state-of-the-art Biotrove OpenArray platform. Different IAC targets were applied at a range of concentrations, yielding a calibration IAC curve for each individual DNA sample. The developed IACs were optimized, tested, and validated by using more than 5,000 unique qPCR amplifications, allowing accurate quantification of microorganisms when applied to soil DNA extracts containing various levels of PCR-inhibiting compounds. To our knowledge, this is the first study using a suite of IACs at different target concentrations to monitor PCR inhibition across a wide target range, thereby allowing reliable and accurate quantification of microorganisms in PCR-inhibiting DNA extracts. The developed IAC is ideally suited for high-throughput screenings of, for example, ecological and agricultural samples on next-generation qPCR platforms.

Microfluidic Quantitative PCR for Simultaneous Quantification of Multiple Viruses in Environmental Water Samples

“…In fact, when RT and qPCR was performed with 10-fold-diluted RNA samples, sample recovery efficiencies were improved from 9.4% to 27.0%, whereas NoV GI and GII and RoV dropped below the quantification limit (data not shown). Regardless, the sample process control and IAC are useful for evaluating the performance for virus concentration, RNA extraction and purification, RT, STA, and qPCR (18,29,47). We can determine if any of these processes need to be redone based on low sample recovery efficiencies or IAC recovery efficiencies.…”

Simultaneous Quantification of Multiple Food- and Waterborne Pathogens by Use of Microfluidic Quantitative PCR

“…Process controls are used to evaluate and normalize the DNA recovery efficiency when processing samples for qPCR (7,48), while internal amplification controls are used to evaluate the DNA quality (e.g., the presence of PCR inhibitors) in each DNA sample (47,49). In this study, we used Pseudogulbenkiania sp.…”

“…Similar DNA recovery efficiencies were obtained from EHEC strain Sakai and strain NH8B, suggesting that strain NH8B could be used as a process control during the quantification of EHEC. Although it was not examined in this study, the NH8B_3641 standard plasmid could also be used as a noncompetitive internal amplification control (49) if added to the sample DNA.…”

“…The inclusion of process controls and internal amplification controls are important for ensuring accurate quantification in qPCR experiments (7,(47)(48)(49). Process controls are used to evaluate and normalize the DNA recovery efficiency when processing samples for qPCR (7,48), while internal amplification controls are used to evaluate the DNA quality (e.g., the presence of PCR inhibitors) in each DNA sample (47,49).…”

Microalgae: Current Research and Applications

“…Secondly, the GeoChip also follows the microarray design concept with 167,044 50-mer oligos covering 395,894 coding sequences from bacteria, achaea, fungi and viruses (He et al,0000). Thirdly, the Biotrove Open Array system has been used extensively for environmental samples (van Doorn et al, 2009). It allows high throughput qPCR reactions in 33 nL volumes.…”