Abstract
Increased attention has been paid to the endosymbiotic bacteria of insects. Because most insect endosymbionts are uncultivable, quantitative PCR (qPCR) is a practical and convenient method to quantify endosymbiont titers. Here we report a protocol for real-time qPCR based on SYBR Green I fluorescence as well as some tips to prevent possible pitfalls.
Keywords: Bacteria, Endosymbionts, Insects, qPCR, Quantification, SYBR Green
Background
Insects often harbor bacterial symbionts of various taxa in their bodies. Such bacterial symbionts (endosymbiotic bacteria) attract great attention because of their profound effects on the host insect. Some bacteria provide essential nutrition to their hosts (Baumann et al., 1995), some confer resistance against parasites (Oliver et al., 2003; Hedges et al., 2008), and some even manipulate reproduction or sex determination of their hosts for their own benefit (Werren et al., 2008; Kageyama et al., 2012). Because most insect endosymbionts are uncultivable, quantitative PCR (qPCR) is a practical and convenient method to quantify endosymbiont titers (Simoncini et al., 2001), possibly complemented by other visualization methods, such as fluorescence in situ hybridization (FISH) (Koga et al., 2009) and/or electron microscopy.
Materials and Reagents
Note: Reagents differ depending on the qPCR equipment. Here I describe a protocol for absolute quantification using LightCycler® 480 (Roche). For each reaction, two or more technical replicates are strongly recommended.
Equipment
Software
Procedure
Data analysis
After each reaction, check the melting curve to confirm that the correct products have been amplified. Samples with cycle threshold (Ct) value of > 35 should be considered uninfected or infected at an undetectable level. Figure 2. Melting curves. Melting curves should be uniform (red lines on the left panel). Two or more patterns (e.g., red and green lines on the right panel) indicate that PCR products are different.
Notes
Recipes
Acknowledgments
This work was supported by KAKENHI ( 16K08106), Japan.
References
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