Fig. 1
General workflow and timeline of the Mycoplasma and Chlamydia assays
Validated protocols for differentiation among 37 Mollicutes species [10] and nine Chlamydia spp. [28] have been published by our group. Other applications based on AS assays include DNA serotyping of Salmonella enteric [29], genotyping of methicillin-resistant Staphylococcus aureus [30], enterohemorrhagic Escherichia coli [31], and many more.
In the present chapter, we describe the procedure for detection of 83 Mollicutes species (Mycoplasma assay, see Table 1) and 11 Chlamydia spp. (Chlamydia assay). Basic principles of assay design and experimental features are given in Notes 2 and 3.
Table 1
Selection of important pathogenic animal and human mycoplasmas and cell culture contaminants that can be identified using the Mycoplasma microarray assay
Animal mycoplasmas | Human mycoplasmas | Mycoplasmas in cell culture |
|---|---|---|
M. agalactiae | M. genitalium | A. laidlawii |
M. bovigenitalium | M. hominis | M. arginini |
M. bovis | M. pneumoniae | M. fermentans |
M. californicum | U. parvum | M. hyorhinis |
M. capricolum subsp. capripneumoniae a | U. urealyticum | M. orale |
M. conjunctivae | M. salivarium | |
M. dispar | ||
M. felis | ||
M. gallisepticum | ||
M. hyopneumoniae | ||
M. mycoides subsp. mycoides a | ||
M. ovipneumoniae | ||
M. suis | ||
M. synoviae |
2 Materials
2.1 DNA Extraction
Commercial DNA extraction kit, e.g., High Pure PCR Template Preparation Kit (Roche Diagnostics, Mannheim, Germany) suitable for cultured strains and a wide range of tissue samples, e.g., nasal, vaginal and conjunctival swabs, mucus, bronchoalveolar lavage, organs, feces, and milk.
2.2 Biotinylation PCR
1.
PCR-grade water.
2.
PCR reagents. We use DyNAmo™ Flash SYBR™ Green qPCR Mastermix (Finnzymes Vantaa, Finland) for pre-hybridization amplification of mycoplasmal DNA and conventional Taq DNA polymerase, e.g., Native Taq Polymerase (Fermentas, St. Leon-Rot, Germany) or Phusion Green High-Fidelity DNA Polymerase (Thermo Scientific, Dreieich, Germany) with the corresponding buffer system and a dNTP mixture (2 mM of each nucleotide) for chlamydial samples.
3.
Primers. The sequences of primer oligonucleotides are given in Table 2. The concentration of stock solutions is 100 pmol/μl and of working solutions 10 pmol/μl.
Table 2
Primers for biotinylation PCR
Name | Sequence 5′–3′a | Target gene | Amplicon size | References |
|---|---|---|---|---|
F1388 | 5′Bio-GTT TCC TGG GCA AGG TTC G-3′ | Mycoplasma | 594 bp | [10] |
23S rDNA | ||||
R1982 | 5′Bio-CCG TTA TAG TTA CGG CCG CC-3′ | |||
tuf-064F | 5′Bio-ATG CCN CAA ACW MGW GAA CAC-3′ | Mycoplasma tuf | 614 bp | [10] |
tuf-681R | 5′Bio-TRT GAC KWC CAC CTT CWT CTT-3′ | |||
U23F-19 | 5′Bio-ATT GAM AGG CGA WGA AGG A-3′ | Chlamydia | 171 bp | [28] |
23S rDNA | ||||
23R-22 | 5′Bio- GCY TAC TAA GAT GTT TCA GTT C-3′ | |||
EGFP-11F | 5′Bio-CAG CCA CAA CGT CTA TAT CAT G-3′ | Internal control | 276 bp | [33] |
EGFP-10R | 5′Bio-CTT GTA CAG CTC GTC CAT GC-3′ |
4.
Internal amplification control pGEM-EGFP2rev [33]. Commercially available as INTYPE IC-DNA from Qiagen Leipzig, Germany.
2.3 Electrophoresis
1.
Agarose, molecular biology grade: 1.5 % (w/v) gels.
2.
Tris-borate EDTA electrophoresis buffer (TBE): 0.09 M of Tris-borate, 0.002 M of EDTA, pH 8.0. For 1 L of 10× TBE, mix 108 g of Tris-base, 55 g of boric acid, and 80 mL of 0.25 M EDTA, make up with water. Dilute 1:10 before use.
3.
Gel loading buffer (GLB): 20 % (v/v) of glycerol, 0.2 M of EDTA, 0.01 % (w/v) of bromophenol blue, 0.2 % (w/v) of Ficoll 400.
4.
Ethidium bromide stock solution: 1 % (10 mg/mL) solution in water. Caution: the substance is presumed to be mutagenic. Avoid direct contact with skin. Wear gloves when preparing solutions and handling gels.
5.
DNA size marker. We mostly use the O’RangeRuler (“100-bp ladder”) (Fermentas, St. Leon-Rot, Germany).
2.4 DNA Microarray Hybridization
The Identibac™ Hybridisation Kit (Alere Technologies, Jena, Germany) is the most efficient and convenient option and contains all necessary reagents: hybridization buffer (C1), washing buffers (C2, C5), streptavidin-peroxidase conjugate (C3 + C4), and TMB substrate (D1). Alternatively, buffers can be prepared manually according to the protocol published previously [32].
2.5 General Equipment and Consumables
1.
Benchtop centrifuge, e.g., 5402 (Eppendorf, Hamburg, Germany).
2.
Vortex shaker, e.g., Vortex 1 (IKA Labortechnik, Staufen, Germany).
3.
Set of pipettes covering the volume range from 0.1 to 1,000 μl and aerosol-resistant filter tips.
4.
Sterile, DNAse and RNAse-free plastic tubes.
5.
Thermal cycler. We use the Mx3000P with MxPro™ 4.10. software (Agilent, Waldbronn, Germany) for Mycoplasma assay and Mastercycler personal (Eppendorf, Hamburg, Germany) for Chlamydia assay.
6.
Apparatus for horizontal gel electrophoresis.
7.
UV transilluminator, 254 nm and/or 312 nm.
8.
Video documentation or photographic equipment.
9.
ArrayStrip™ or ArrayTube™ reaction vessels (Alere Technologies, Jena, Germany) with integrated DNA microarray chips for the Mycoplasma assay (layout Myc_05 or higher) or the Chlamydia assay (layout Chlam_gesamt_4_AS or higher). These arrays are commercially available from Alere. The company also has a service for ordering customized arrays. The sequences of the oligonucleotide probes immobilized on these arrays were previously published in Sachse et al. [32] (Chlamydia assay) and Schnee et al. [10] (Mycoplasma assay).
10.
Heatable horizontal tube shaker. We recommend the BioShake iQ (Quantifoil Instruments, Jena, Germany; see Note 4).
11.
ArrayMate transmission reader (Alere Technologies, Jena, Germany).
12.
Iconoclust software, version 2.3 or higher (Alere Technologies, Jena, Germany).
3 Methods
3.1 DNA Extraction
Follow the protocol given by the commercial supplier of the DNA extraction kit.
3.2 Pre-hybridization Biotinylation PCR
3.2.1 Mycoplasma Assay
The two genomic targets in the 23S rRNA and tuf genes are amplified independently in two separate real-time PCRs. The former is amplified in a simplex reaction, and the latter is co-amplified with the internal control template, i.e., plasmid pGEM-EGFP2rev [33] (see Note 5).
1.
Prepare master mixtures according to Table 3 as a multiple of 20 μl depending on sample numbers.
Table 3
Composition of the reaction mixtures for pre-hybridization real-time PCR in the Mycoplasma assay
Target | Components | Working concentration | Volume per reaction (total 20 μl) | Final concentration |
|---|---|---|---|---|
23S rDNA | PCR-grade water | – | 7 μl | – |
DyNAmoq PCR Mastermix | 2× | 10 μl | 1× | |
Primer F1388 | 10 pmol/μl | 1 μl | 500 nM | |
Primer R1982 | 10 pmol/μl | 1 μl | 500 nM | |
DNA template | – | 1 μl | – | |
tuf | PCR-grade water | – | 5.9 μl | – |
DyNAmoq PCR Mastermix | 2× | 10 μl | 1× | |
Primer tuf-064F | 10 pmol/μl | 1 μl | 500 nM | |
Primer tuf-681R | 10 pmol/μl | 1 μl | 500 nM | |
ctrl primer EGFP-11F | 10 pmol/μ | 0.5 μl | 250 nM | |
ctrl primer EGFP-10R | 10 pmol/μl | 0.5 μl | 250 nM | |
IC: pGEM-EGFP2reva | 105 copies/μl | 0.1 μl | 104 copies/rxn | |
DNA template | – | 1 μl | – |
2.
Add template to each reaction vessel: 1 μl of DNA extract from your sample. If the DNA contents of the extract is low, up to 4 μl of sample DNA can be used and the amount of water be reduced accordingly.
3.
In each series, include external amplification controls that contain DNA of mycoplasma reference strains, e.g., M. bovis PG45, as positive control, and water, instead of sample extract, as negative control.
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