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

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Detection of L. monocytogenes Using PCR 187 Fig. 1. (A) Reverse hybridization detection of a biotinylated PCR product. The probe on the membrane specifically captures the biotinylated PCR product. The probe product hybrid is then detected via a streptavidin alkaline phosphate conjugate and conversion of the substrate to an insoluble product, yielding a purple color on the membrane. (B) Actual Biodyne C membrane strip following colorimetric detection using an L. monoctogenes-specific probe in the membrane. Also on the strip is the positive detection control PosDet and the unrelated negative control.
188 O'Connor 2.2. PCR of Enriched Food Samples 1. Oligonucleotide primers: LGS2 5'-ccgtgcgccctttctaactt-3', LMONOREV 5'-tttgttcagttttgagaggt-3' (Genosys Biotechnologies, Ltd., London Road, Pampisford, Cambridgeshire). 2. Nuaire 425 Class II Biological Safety Cabinet. 3. Thermal cycler. 4. PCR tubes, 0.5 mL. 5. PCR-grade H2O (Sigma UK). 6. Proteinase K (10 mg/mL) (Sigma). 7. Taq DNA polymerase, MgCl2, and reaction buffer (Promega, Madison, WI, USA). 8. DU:dNTPs (Promega): Prepare from 100 mM stocks by combining 12.5 µL dATP, dGTP, dCTP, 4.45 µL dTTP, and 8 µL dUTP. 9. Uracil-N-DNA glycosylase (UNG) (Roche Molecular Biochemicals, UK) 2.3. Reverse Hybridization Assay 1. Biodyne C membrane (Pall Biosupport; Pall Europe, Ltd. Portsmouth UK). 2. EDAC-HCl (Sigma) 16% (w/v) in distilled water (dH 2O). 3. Oligonucleotide probes (LMONO20 5'-gatgcttcaaggcatagtgcc-3'), including PosDet a positive detection control probe (Genosys Biotechnologies Ltd.). PosDet consists of a random stretch of oligonucleotide bases modified with a biotin group at the 3'-end and an amine group at the 5'-end. 4. NaHCO 3: 0.5 M, pH 8.4. 5. Biodot liquid dispensing machine (xyz 3000) (Biodot, Irvine, CA, USA). 6. NaOH: 0.1 M. 7. Biodot cutter 3000. 8. Heating block. 9. Hybridization buffer: 5X SSPE (20X SSPE: 175.3 g NaCl, 27.6 g NaH 2PO 4·H 2O, 7.4 g EDTA/L H 2O), 0.1% (w/v) sodium dodecyl sulfate (SDS). 10. TBS: 50 mM Tris-HCl, pH 7.5, 0.15 M NaCl. 11. Shaking platform. 12. Strepatvidin alkaline phosphatase conjugate (Calbiochem-Novabiochem UK, Ltd., Nottingham, UK). 13. TBS/Tween ® 20: 50 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 1% Tween. 14. Color development solution: 0.1 M Tris-HCl, pH 9.5, 50 mM MgCl 2, 0.1 M NaCl. 15. Chromogenic substrates: nitroblue tetrazolium chloride (NBT) and 5-bromo- 4-chloro-3-indolyl-phosphate, 4-toluidine salt (BCIP) (Roche Molecular Biochemicals). To 10 mL of color development solution, 50 µL of NBT and 37.5 µL BCIP are added. 3. Methods 3.1. Enrichment of Food Samples (see Notes 1 and 2) 1. Weigh out 25 mL/25 g of sample. 2. Place sample on sterile stomacher bag and add 225 mL of enrichment broth. 3. Homogenize the sample, seal the top of the bag, and incubate at 37°C overnight.
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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
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 and 162: 164 Gallien The first description o
Page 163 and 164: 166 Gallien
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
Page 177 and 178: 180 Gallien Fig. 3. Photographic im
Page 179 and 180: 182 Gallien 3. Boyce, T. G., Swerdl
Page 181 and 182: 184 Gallien enteropathogenic E. col
Page 183: 186 O'Connor This chapter will desc
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
Page 195 and 196: Fig. 1. Sensitivity of the PCR assa
Page 197 and 198: 200 Lester and LeFebvre 5. Swart, K
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
Page 209 and 210: 212 Skuce et al. 2. Carefully trans
Page 211 and 212: Table 4 Recommended PCR Amplificati
Page 213 and 214: 216 Skuce et al. Fig. 3. PCR produc
Page 215 and 216: 218 Skuce et al. Fig. 5. Spoligotyp
Page 217 and 218: 220 Skuce et al. 14. Aranaz, A., Li
Page 219 and 220: 222 Skuce et al.
Page 221 and 222: 224 Khan Simultaneous detection of
Page 223 and 224: 226 Khan 10. Incubate for 20 min at
Page 225 and 226: 228 Khan 5. Periodically, multiplex
Page 227 and 228: 230 Khan
Page 229 and 230: 232 Hotzel et al. tis (2). Particul
Page 231 and 232: 234 Hotzel et al. 8. Sodium dodecyl
Page 233 and 234: 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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PCR Detection of Microbial Pathogens PCR Detection of Microbial ...

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