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

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STEC PCR 165 Fig. 1. Routes of transmission of STEC to humans The eae gene (with subtypes α,β,γ,δ, and ε) coding for a protein called intimin completes a key function as it effects a closer connection between pathogen and host cell. A pedestrial cup formed beneath the bacterial cell is built by the effacing of microvilli so that the bacterial cell is able penetrate into the host. The HPI also comprises a gene cluster. Some proteins derived from this locus are involved in iron uptake from the nutrient media or the host. The ast A gene was found in enteroaggregative E. coli. The gene product is a heat-stable toxin, which may contribute to the disturbance of the electrolyte balance of host cells. Virulence factors located on plasmids include the EHEC-hemolysin (EHEChly) (33–36), the kat P gene, which encodes an enzyme with catalase-peroxidase-activity (37), the esp P gene encoding a serin protease cleaving coagulation factor V of human blood (38), the etp gene cluster encoding proteins of a type II secretion system (39), the col D 157 gene involved in colicin production (40), and the ile X gene, a tRNA gene related to stx 2 and subtypes only (41). In the present chapter, a cascade of methods for specific detection, isolation, and characterization of STEC from different habitats and sample matrixes is described. The general course of the procedure and individual steps involved are summarized in Fig. 2 (42,43).
166 Gallien
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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
Page 111 and 112: 114 Englen et al. 3.1.3. Fecal Samp
Page 113 and 114: 116 Englen et al. respectively), an
Page 115 and 116: 118 Englen et al. Fig. 1. Identific
Page 117 and 118: 120 Englen et al. 3. Goossens, H.,
Page 119 and 120: 122 Englen et al.
Page 121 and 122: 124 Sachse and Hotzel Table 1 Compa
Page 123 and 124: 126 Sachse and Hotzel Table 2 Prime
Page 125 and 126: 128 Sachse and Hotzel 7. Phenol: sa
Page 127 and 128: 130 Sachse and Hotzel 4. Vortex mix
Page 129 and 130: 132 Sachse and Hotzel Fig. 2. Speci
Page 131 and 132: 134 Sachse and Hotzel In the latter
Page 133 and 134: 136 Sachse and Hotzel 17. Longbotto
Page 135 and 136: 138 Popoff spastic paralysis. The g
Page 137 and 138: Table 3 Primers for Toxin Gene Dete
Page 139 and 140: 142 Popoff 18. Agarose gel loading
Page 141 and 142: 144 Popoff Taq reaction buffer 1X,
Page 143 and 144: 146 Popoff almost all C. perfringen
Page 145 and 146: 148 Popoff 2. Add to each PCR tube
Page 147 and 148: 150 Popoff 7. Hielm, S., Hyyttia, E
Page 149 and 150: 152 Popoff 35. Wieckowski, E., Bill
Page 151 and 152: 154 Berri et al. 2. Materials 1. C.
Page 153 and 154: 156 Berri et al. 3.3.2. Vaginal Swa
Page 155 and 156: 158 Berri et al. Fig. 1. Trans-PCR.
Page 157 and 158: 160 Berri et al. Fig. 3. Sensitivit
Page 159 and 160: 162 Berri et al.
Page 161: 164 Gallien The first description o
Page 165 and 166: 168 Gallien 2. Materials 2.1. Bacte
Page 167 and 168: 170 Gallien 10. Ethidium bromide: 1
Page 169 and 170: Table 2 Components (in µL) of 25-
Page 171 and 172: Table 4 Components (in µL) of 25-
Page 173 and 174: 176 Gallien 3.3. Specific Isolation
Page 175 and 176: Table 6 PCR Conditions for Subtypin
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Page 181 and 182: 184 Gallien enteropathogenic E. col
Page 183 and 184: 186 O'Connor This chapter will desc
Page 185 and 186: 188 O'Connor 2.2. PCR of Enriched F
Page 187 and 188: 190 O'Connor 4. Notes 1. In enrichi
Page 189 and 190: 192 O'Connor 4. O’Sullivan, N., F
Page 191 and 192: 194 Lester and LeFebvre titers (4).
Page 193 and 194: 196 Lester and LeFebvre 3.1.2. Samp
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Page 199 and 200: 202 Skuce et al. Table 1 Significan
Page 201 and 202: 204 Skuce et al. commercial kits. E
Page 203 and 204: 206 Skuce et al. Fig. 1. Schematic
Page 205 and 206: 208 Skuce et al. 5. Disposable ster
Page 207 and 208: 210 Skuce et al. 2.5.2. Sequence An
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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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Table 2 Oligonucleotide Primers for
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264 Christensen et al. 3.2. Extract
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Table 3 PCR Protocols for the Patho
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268 Christensen et al. 2. Apply fra
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270 Christensen et al. 7. To reduce
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272 Christensen et al. 24. Fisher,
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274 Christensen et al. 54. Hunt, M.
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276 Malorny and Helmuth valent meta
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278 Malorny and Helmuth 12. Apparat
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280 Malorny and Helmuth 3.3. Amplif
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282 Malorny and Helmuth Table 2 Vol
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284 Malorny and Helmuth Table 4 PCR
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286 Malorny and Helmuth 3. Wallace,
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288 Malorny and Helmuth
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290 Wastling and Mattsson is an imp
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292 Wastling and Mattsson 2. Remove
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294 Wastling and Mattsson Fig. 1. B
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296 Wastling and Mattsson time PCR
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298 Wastling and Mattsson
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300 Pozio and La Rosa Table 1 Princ
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302 Pozio and La Rosa regions; spot
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304 Pozio and La Rosa 3. Proteinase
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306 Pozio and La Rosa Table 3 PCR-R
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308 Pozio and La Rosa 2. Pepsin sho
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310 Pozio and La Rosa
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312 Knutsson and Rådström The ref
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314 Knutsson and Rådström Fig. 1.
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316 Knutsson and Rådström 2.4. El
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318 Knutsson and Rådström the swa
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320 Knutsson and Rådström Fig. 3.
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322 Knutsson and Rådström 4. Alek
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324 Knutsson and Rådström 34. Hee
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