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

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Table 4 Primers for Toxin Gene Detection in other Clostridium spp. T°C Accession Toxin Primer Sequence (5' —> 3') Positions a of annealing Size(bp) number ToxA C. difficile P533 CCT AAT ACA GCT ATG GGT GCG AAT GG 7919–7944 M30307 ToxA C. difficile P534 GGG GCT TTT ACT CCA TCA ACA CCA AAG 8251–8277 R 55 358 ToxB C. difficile P537 AAA AAT GGA GAG TCA TTC AAC 1137–1157 X53138 ToxB C. difficile P538 GCC CTT GAT TTA TAA TAC CC 1538–1557 R 55 420 CDTa C. difficile P368 GAA GCA GAA AGA ATA GAG C 312–331 L76081 CDTa C. difficile P589 GGT TTT TCA TCA CCT TTT CCA GG 675–697 R 55 385 CDTb C. difficile P304 TAA ACA AAG GAG AAT CTG C 2680–2699 L76081 CDTb C. difficile P590 TTT CTA ATT TAA TTT GCT TTC CAG C 2953–2977 R 55 297 Tox α novyi P531 GAA GGA GAT AAA AGT GCT ATA AAT TAT AAA GG 6146–6177 Z48636 Tox α novyi P532 CAC CTA ATA CTC GCC AAC CCG TTA CTG CAC 6555–6585 R 55 439 Tox α septicum P512 CTT ACA AAT CTT GAA GAG GGG GG 654–676 D17668 Tox α septicum P513 CAT TTG GAT TGT ATC TAG CAG 924–904 R 55 239 LT C. sordellii P535 CTA GAA AAA TTT GCT GAT GAG GAT TTG GTA AG 1202–1233 X82638 LT C. sordellii P536A GG CTT GAT TTA TAA CAG AGT TAT TGG C 1607–1634 R 55 432 Neuramnidase C. sordellii P708 GACTTTGGCAGATGGTACTATGCTAGC 180–206 U77593 Neuramnidase C. sordellii P709 CATCAGAATAAACCATTTGAACAGACC 458–481R 55 301 rRNA gene C. chauvoei CC16S-L GTCGAGCGAGGAGAGTTC 54–71 58 960 M59091 rRNA gene C. chauvoei CC16S-R TCATCCTGTCTCCGAAGA 996–1013R Tetanus toxin C. tetani P476 ATGCCAATAACCATAAATAATTTTAGATATAG 397–P292 55 1475 X04436 Tetanus toxin C. tetani P477 TTCATCTTGAAATGGTTCTTCTG 1734–1756R a R indicates reverse primer. Clostridium PCR 141
142 Popoff 18. Agarose gel loading dye: 100 mM EDTA, 30% glycerol, 0.25 % bromophenol blue. 19. Denaturing solution: 200 mM NaOH, 500 mM EDTA. 20. Hybridization buffer: 5X saline sodium citrate (22 g/L sodium citrate, 43.8 g/L NaCl), 0.1% laurylsarcosine sodium, 0.02% sodium dodecyl sulfate, 1% blocking reagent (Roche Diagnostics, Penzberg, Germany). 21. Washing buffer: 100 mM Tri-HCl, pH 7.5, 150 mM NaCl, 0.5 g/L Tween 20 ® , 1% blocking reagent, 100 µg/mL fish sperm DNA. 22. Revelation buffer: 100 mM maleic acid, pH 7.5, 150 mM NaCl, 1% blocking reagent, 1% peroxidase-labeled anti-digoxigenin antibody (Roche Diagnostics). 23. 3,3',5,5'-tetramethylbenzidine (Roche Diagnostics). 24. Stop solution: 1.5 M H 2SO 4. 3. Methods 3.1. Cultures 3.1.1. Broth Medium Inoculate one colony from an agar medium culture in 10 mL of broth medium. 3.1.2. Agar Medium Use an isolated colony from TGY agar medium or other solid medium for anaerobic bacteria for PCR detection. 3.1.3. Enrichment Culture from Food or Biological Samples Food: Inoculate 25 g in 225 mL of TGY broth. Biological samples: The content from small intestine is the preferred sample for diagnosis of clostridial gastro intestinal diseases. Use muscle or exudation from gangrenous lesions. Inoculate 1 mL or 1 g in 9 mL of TGY broth. Culture in anaerobic conditions at 37°C for 18 h. Anaerobic jars containing gaspack or H 2-CO 2 (95:5), or an anaerobic chamber can be used. 3.2. DNA Extraction 3.2.1. DNA Extraction from Broth Culture 1. Centrifuge 1 mL culture for 10 min at 13,000g. 2. Wash the pellet in 1 mL of distilled water. 3. Suspend the pellet in 200 µL of Instagen. 4. Incubate at 56°C for 30 min and vortex mix vigorously for 10 s. 5. Incubate for 10 min at 95°C and vortex mix vigorously for 10 s. 6. Centrifuge for 10 min at 13,000g. The supernatant is immediately used for PCR. 3.2.2. DNA Extraction from Colonies Grown on Agar Medium Resuspend one colony in 200 µL of Instagen and proceed with the Instagen protocol as above (see Subheading 3.2.1., steps 4–6).
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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
Page 87 and 88: 90 Frey 13. Lysis buffer: 100 mM Tr
Page 89 and 90: 92 Frey as template for PCR. In add
Page 91 and 92: 94 Frey strains, including all sero
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Page 97 and 98: 100 Ewalt and Bricker 4. Ethidium b
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Page 105 and 106: 108 Ewalt and Bricker unused ethidi
Page 107 and 108: 110 Englen et al. which campylobact
Page 109 and 110: 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: Table 3 Primers for Toxin Gene Dete
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 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
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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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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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