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Quantification of Densities of Bacterial Endosymbionts of Insects by Real-time PCR

实时PCR定量分析昆虫内共生细菌的密度

Daisuke Kageyama Daisuke Kageyama

发布: 2017年10月05日第7卷第19期 DOI: 10.21769/BioProtoc.2566 浏览次数: 10793

评审: Filipa VazModesto Redrejo-RodriguezAnonymous reviewer(s)

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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 (细菌) search Endosymbionts (内共生体) search Insects (昆虫) search qPCR (qPCR) search Quantification (定量) search SYBR Green (SYBR Green) search

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.

  1. Pipette tips:
    10-μl tips (e.g., Fukaekasei and Watson, catalog number: 110-201C )
    200-μl tips (Fukaekasei and Watson, catalog number: 1201-705C )
    1,000-μl tips (Fukaekasei and Watson, catalog number: 110-706C )
    To reduce the risk of contamination, use of the filtered tips, e.g.,
    10-μl tips (Fukaekasei and Watson, catalog number: 1251-204CS )
    200-μl tips (Fukaekasei and Watson, catalog number: 1252-703CS )
    1,000-μl tips (Fukaekasei and Watson, catalog number: 124-1000S )] is preferable
  2. Centrifuge tubes of 1.5 ml (e.g., Fukaekasei and Watson, catalog number: 131-7155C )
  3. Centrifuge tubes of 50 ml [e.g., Falcon 50-ml conical centrifuge tubes (Corning, Falcon®, catalog number: 352070 )]
  4. LightCycler® 480 Multiwell Plate 96, white (Roche Molecular Systems, catalog number: 04729692001 , sealing foils included)
  5. Template DNA to be measured (genomic DNA extracted from insects)
  6. Template DNA for standard (PCR products or inserted plasmid DNA with known concentration)
  7. LightCycler® 480, buffer 1 (2x Master mix) (Roche Molecular Systems, catalog number: 04707516001 )
  8. LightCycler® 480, buffer 2 (H2O) (Roche Molecular Systems, catalog number: 04707516001 )
  9. Forward and reverse primers
  10. Reaction mix (see Recipes)

Equipment

  1. Spectrophotometer (Bio-Rad Laboratories, model: SmartSpecTM 3000 )
  2. LightCycler® 480 System (Roche Molecular Systems, model: LightCycler® 480 ) connected to a computer
  3. Centrifuge for 96-well plate (e.g., Kubota, model: PlateSpin II )
  4. Pipettes [e.g., PIPETMAN® P10 (Gilson, catalog number: F144802 ), P20 (Gilson, catalog number: F123600 ), P100 (Gilson, catalog number: F123615 ), P200 (Gilson, catalog number: F123601 ), or P1000 (Gilson, catalog number: F123602 )]
  5. Laminar flow cabinet

Software

  1. LightCycler® 480 Software, version 1.5.1 (Roche Molecular Systems)
  2. Primer3Plus free online software (http://primer3plus.com/)

Procedure

English
中文翻译

文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Kageyama, D. (2017). Quantification of Densities of Bacterial Endosymbionts of Insects by Real-time PCR. Bio-protocol 7(19): e2566. DOI: 10.21769/BioProtoc.2566.

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分类

微生物学 > 微生物-宿主相互作用 > 细菌

微生物学 > 体内实验模型 > 细菌

分子生物学 > DNA > PCR

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