Abstract
With PCR becoming one of the most important and widely-used diagnostic tools for infectious diseases of poultry, an urgent need has developed for an endogenous internal control (EIC) that monitors the quality and quantity of poultry DNA in test samples. In this study we developed a SYBR-qPCR to target the poultry homolog of the hydroxymethylbilane synthase (HMBS) gene as an EIC for avian species. The avian HMBS-based qPCR was very sensitive, detecting one HMBS gene copy in a 20 µL reaction, and is highly specific for avian species. It amplified DNA from 11 organs and tissues of chickens showing it can be used as an EIC on a large variety of samples. The application of the established EIC on clinically and experimentally infected samples demonstrated that false negativity and result variations could result from samples being collected using different operators, techniques, preservatives, and storage times. The high sensitivity and specificity of the avian HMBS-based qPCR, its ability to quantify DNAs extracted from a wide range of tissues and poultry species along with its usefulness in reducing false negativity in PCR results associated with inadequate sampling and storage degradation makes it an ideal EIC for poultry DNA and RNA PCR diagnostics. The study also highlights the importance of appropriate sampling and storage of samples in ensuring accuracy of molecular diagnostic testing.
Highlights
Due to its exquisite sensitivity and specificity, polymerase chain reaction (PCR) has become one of the most valuable, powerful and widely-used tools for rapid detection of infectious agents in poultry (Okamatsu et al 2016; Stoute et al 2019; Hussein et al 2019; de Wit et al 2018; Kaltenboeck et al 2005; Luan et al 2016)
Establishment of the avian hydroxymethylbilane synthase (HMBS)‐based qPCR The BLASTn program showed that the sequences of the primers selected for the avian HMBS-based qPCR were highly specific and recognized all the bird species we intended to study (Fig. 2)
The avian HMBS-based qPCR was further verified when it amplified the DNAs from a duck, a goose, a pigeon, seven indigenous chicken species of China, and four rare bird species
Summary
Due to its exquisite sensitivity and specificity, polymerase chain reaction (PCR) has become one of the most valuable, powerful and widely-used tools for rapid detection of infectious agents in poultry (Okamatsu et al 2016; Stoute et al 2019; Hussein et al 2019; de Wit et al 2018; Kaltenboeck et al 2005; Luan et al 2016). To monitor the efficiency of DNA extraction from samples, its concentration and degree of degradation, and the PCR operation itself, various exogenous and endogenous internal controls (IC) have been established (Fig. 1). The exogenous IC is only applied to the test samples after they have been collected, stored and transported to a laboratory for processing. They do not evaluate these important phases of the process (Fig. 1). Endogenous internal controls (EIC) are not associated with these problems as they are reference genes of the host already present in the test sample. Any damage to the sample at any stage will interfere with the EIC and alert the operator that PCR results might be inaccurate and need further scrutiny
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