PCR Detection of Microbial Pathogens PCR Detection of Microbial ...

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Yersinia PCR 313 recently been developed to improve high-throughput PCR and the diagnosis of Y. enterocolitica (30). The medium was developed through a mathematical approach by the use of screening factorial design experiments and confirmatory tests. Factors studied included PCR inhibition, growth of Y. enterocolitica, and growth of background flora (see Fig. 1). The formulation of the PCR-compatible enrichment medium is based on a number of requisites: (i) the buffer system of the medium should mimic the buffer system in the PCR mixture; (ii) neither the medium components nor the metabolites produced by bacterial growth should be PCR-inhibitory; and (iii) selective components must be included to restrict the growth of competing background flora. The YPCE medium has been combined with a multiplex PCR assay that targets two genes, namely, the chromosomal 16S rRNA gene (for species identification) and the plasmid-borne virulence gene yadA (for virulence confirmation) (29). This chapter describes a simple and rapid PCR protocol for the diagnosis of pathogenic pYV Y. enterocolitica strains for veterinary use. An overview is presented in Fig. 2. 2. Materials 2.1. Swab Sampling 1. A wooden-stemmed cotton-tipped swab (15 cm) (SelefaTrade, Spånga, Sweden). 2. Swab sample dilution fluid (NaCl-Peptone-Water). 2.1.1. Preparation of Swab Sample Dilution Fluid (NaCl-Peptone-Water) Add all the ingredients to a suitable flask: 1.0 g Peptone (Merck, Darmstadt, Germany), 8.5 g NaCl, and 1 L distilled water. The pH should be around 7.0 ± 0.1, adjust if necessary. Sterilize the dilution fluid at 121°C for 15 min. Store the dilution fluid at 0–5°C until required. 2.2. Enrichment in YPCE Medium 1. 15-mL plastic test tubes (Sarstedt, Nümbrecht, Germany). 2. 10-mL YPCE medium. 2.2.1. Preparation of YPCE Medium 1. Add the following ingredients to a suitable flask: 10g Tryptone (peptone from casein, pancreatically digested) (Merck, Cat. no. 1.07213), 2.75 g MOPS (3-[Nmorpholino] propanesulfonic acid) (Sigma, St Louis, MO, USA), 2.75 g Tris buffer (ICN Biochemicals, Costa Mesa, CA, USA), 20.0 g D-sorbitol (Sigma), and 1 L distilled water.
314 Knutsson and Rådström Fig. 1. Illustration of the development of the medium, which was based on screening and factorial design experiments, to find a medium composition that fulfills the requirements for a PCR-compatible enrichment medium. The medium was designed to optimize the growth of Y. enterocolitica and to minimize the presence of PCRinhibitory components and growth of background flora. Autoclave at 121°C for 15 min and allow to cool to room temperature (25°C). Transfer 983 mL aseptically to a flask of suitable size and add the following ingredients: 2. 2 vials Yersinia-selective-supplement (cefsulodin-irgasan-novobiocin, [CIN]) (Merck, Cat. no. 1.16466.001). The lyophilizate should be suspended in the vial by the addition of 1 mL sterile distilled water. 3. 5 mL sterilized potassium chlorate solution: 10 g KClO 3 (Merck) dissolved in 100 mL H 2O. 4. Finally, adjust the pH to 8.2 ± 0.1 by the addition of 1 M potassium hydroxide (KOH) solution (approx 10 mL). 2.3. PCR 1. PCR test tubes, 0.2-mL MicroAmp reaction tubes with cap (Applied Biosystems, Foster City, CA, USA). 2. Taq DNA polymerase, 5 U/µL (Roche Diagnostics, Mannheim, Germany). 3. 10x PCR buffer (Roche Diagnostics). 4. Primers; Y1, Y2, P1, and P2 (Scandinavian Gene Synthesis, Köping, Sweden). Store stock solution 10 µM of each primer in aliquots at –20°C. 5. Nucleotides; dATP, dCTP, dGTP and dTTP (Roche Diagnostics) and the 10 mM dNTP mixture is stored in aliquots at –20°C.
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TM Methods in Molecular Biology VOL
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4 Sachse 2. Selection of Target Seq
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Table 1 Target Sequences Used in PC
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8 Sachse Fig. 1. Structural organiz
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10 Sachse proteins (66-68), invasio
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12 Sachse Since there appears to be
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14 Sachse In the context of identif
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16 Sachse to partially compensate t
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18 Sachse nostic potential of PCR.
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20 Sachse product has to be confirm
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22 Sachse 7. van Camp, G., Fierens,
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24 Sachse 33. Rappelli, P., Are, R.
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26 Sachse 60. Furrer, B., Candrian,
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28 Sachse 89. Bloch, W. (1991) A bi
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30 Sachse
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32 Rådström et al. Fig. 1. Illust
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34 Rådström et al. immunoglobulin
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36 Rådström et al. and the captur
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Table 2 Comparison of the Performan
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40 Rådström et al. explain these
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42 Rådström et al. 5.1. Proteins
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44 Rådström et al. neous. Consequ
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46 Rådström et al. 11. Powell, H.
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48 Rådström et al. 39. Katcher, H
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50 Rådström et al. 73. Nagai, M.,
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52 Hoorfar and Cook 2. FOOD-PCR: A
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54 Hoorfar and Cook Fig. 2. Practic
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56 Hoorfar and Cook Fig. 4. The str
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Table 58 3 Hoorfar and Cook Test Co
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60 Hoorfar and Cook The choosing an
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62 Hoorfar and Cook 10. Demanding L
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64 Hoorfar and Cook 15. Anon. (1995
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66 Lübeck and Hoorfar ods for the
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68 Lübeck and Hoorfar amplificatio
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70 Lübeck and Hoorfar It may be ne
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72 Lübeck and Hoorfar specificity
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74 Lübeck and Hoorfar The latter i
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76 Lübeck and Hoorfar A simple che
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78 Lübeck and Hoorfar 13. Hanai, K
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80 Lübeck and Hoorfar 43. Mitchell
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82 Lübeck and Hoorfar 70. Lawrence
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84 Lübeck and Hoorfar 98. Lantz, P
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88 Frey Table 1 Presence of the Var
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90 Frey 13. Lysis buffer: 100 mM Tr
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92 Frey as template for PCR. In add
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94 Frey strains, including all sero
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96 Frey
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98 Ewalt and Bricker (named for the
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100 Ewalt and Bricker 4. Ethidium b
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102 Ewalt and Bricker Primer Cockta
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104 Ewalt and Bricker Fig. 2. Typic
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106 Ewalt and Bricker with betaine
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108 Ewalt and Bricker unused ethidi
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110 Englen et al. which campylobact
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112 Englen et al. 18. Dehydrated cu
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114 Englen et al. 3.1.3. Fecal Samp
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116 Englen et al. respectively), an
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118 Englen et al. Fig. 1. Identific
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120 Englen et al. 3. Goossens, H.,
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122 Englen et al.
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124 Sachse and Hotzel Table 1 Compa
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126 Sachse and Hotzel Table 2 Prime
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128 Sachse and Hotzel 7. Phenol: sa
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130 Sachse and Hotzel 4. Vortex mix
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132 Sachse and Hotzel Fig. 2. Speci
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134 Sachse and Hotzel In the latter
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136 Sachse and Hotzel 17. Longbotto
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138 Popoff spastic paralysis. The g
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Table 3 Primers for Toxin Gene Dete
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142 Popoff 18. Agarose gel loading
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144 Popoff Taq reaction buffer 1X,
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146 Popoff almost all C. perfringen
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148 Popoff 2. Add to each PCR tube
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150 Popoff 7. Hielm, S., Hyyttia, E
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152 Popoff 35. Wieckowski, E., Bill
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154 Berri et al. 2. Materials 1. C.
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156 Berri et al. 3.3.2. Vaginal Swa
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158 Berri et al. Fig. 1. Trans-PCR.
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160 Berri et al. Fig. 3. Sensitivit
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162 Berri et al.
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164 Gallien The first description o
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166 Gallien
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168 Gallien 2. Materials 2.1. Bacte
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170 Gallien 10. Ethidium bromide: 1
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Table 2 Components (in µL) of 25-
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Table 4 Components (in µL) of 25-
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176 Gallien 3.3. Specific Isolation
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Table 6 PCR Conditions for Subtypin
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180 Gallien Fig. 3. Photographic im
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182 Gallien 3. Boyce, T. G., Swerdl
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184 Gallien enteropathogenic E. col
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186 O'Connor This chapter will desc
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188 O'Connor 2.2. PCR of Enriched F
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190 O'Connor 4. Notes 1. In enrichi
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192 O'Connor 4. O’Sullivan, N., F
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194 Lester and LeFebvre titers (4).
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196 Lester and LeFebvre 3.1.2. Samp
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Fig. 1. Sensitivity of the PCR assa
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200 Lester and LeFebvre 5. Swart, K
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202 Skuce et al. Table 1 Significan
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204 Skuce et al. commercial kits. E
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206 Skuce et al. Fig. 1. Schematic
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208 Skuce et al. 5. Disposable ster
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210 Skuce et al. 2.5.2. Sequence An
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212 Skuce et al. 2. Carefully trans
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Table 4 Recommended PCR Amplificati
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216 Skuce et al. Fig. 3. PCR produc
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218 Skuce et al. Fig. 5. Spoligotyp
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220 Skuce et al. 14. Aranaz, A., Li
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222 Skuce et al.
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224 Khan Simultaneous detection of
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226 Khan 10. Incubate for 20 min at
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228 Khan 5. Periodically, multiplex
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230 Khan
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232 Hotzel et al. tis (2). Particul
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234 Hotzel et al. 8. Sodium dodecyl
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236 Hotzel et al. 3. Methods 3.1. D
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238 Hotzel et al. 3.1.5. Semen (see
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240 Hotzel et al. Fig. 1 Detection
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242 Hotzel et al. 4. Notes 1. The u
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244 Hotzel et al. in Mycoplasma Pro
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246 Hotzel et al.
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248 Kobisch and Frey PCR assay to d
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250 Kobisch and Frey Table 1 Oligon
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252 Kobisch and Frey 3.2.1. Prepara
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254 Kobisch and Frey First round of
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256 Kobisch and Frey References 1.
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258 Christensen et al. thrombotic m
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260 Christensen et al. Table 1 Spec
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Page 271 and 272: 274 Christensen et al. 54. Hunt, M.
Page 273 and 274: 276 Malorny and Helmuth valent meta
Page 275 and 276: 278 Malorny and Helmuth 12. Apparat
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Page 281 and 282: 284 Malorny and Helmuth Table 4 PCR
Page 283 and 284: 286 Malorny and Helmuth 3. Wallace,
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Page 287 and 288: 290 Wastling and Mattsson is an imp
Page 289 and 290: 292 Wastling and Mattsson 2. Remove
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Page 295 and 296: 298 Wastling and Mattsson
Page 297 and 298: 300 Pozio and La Rosa Table 1 Princ
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Page 301 and 302: 304 Pozio and La Rosa 3. Proteinase
Page 303 and 304: 306 Pozio and La Rosa Table 3 PCR-R
Page 305 and 306: 308 Pozio and La Rosa 2. Pepsin sho
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Page 319 and 320: 322 Knutsson and Rådström 4. Alek
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