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

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PCR for Veterinary Mycobacteria 215 7. PCR product size can be estimated by comparison with molecular weight markers. 8. The E-Gel is photographed using a digital camera, recorded as a digital image (e.g., see Fig. 3) and manipulated using appropriate image analysis software; we use Corel Photo Paint. 3.4.2. Sequence Analysis 1. 16S PCR products are quantified using PicoGreen double-stranded DNA quantitation kit. 2. Appropriate quantities of unpurified PCR products are sent for commercial double-strand sequence analysis. 3. Consensus sequence (see Fig. 4), using the double-strand sequence data, is facilitated by DNASIS software and compared to DNA sequence databases for sequence identification, e.g., GenBank ® (39), European Molecular Biology Laboratory (EMBL) (40), and Ribosomal Databases Project II (41) databases. 3.4.3. Membrane-Based Analysis by Spoligoblot (22) 1. Add 20 µL of Dra/b amplified PCR products to 150 µL of 2X SSPE/0.1% SDS. 2. Heat denature for 10 min at 100°C 3. Transfer immediately to an ice bath. 4. Apply 170 µL to spoligo membrane and develop according to manufacturer’s instructions (e.g., see Fig. 5). 4. Notes 1. Controls. Three sets of controls are used to monitor the pretreatment, sequence capture and PCR amplification stages of the methodology. Preferred pretreatment controls include previously culture and/or PCR positive and negative tissues. For sequence capture, an aliquot of appropriate reference culture is recommended as a positive control and TES buffer only as a negative control. Preferentially, more than one negative control should be used especially when processing several specimens (10). The negative controls should be interspersed throughout processing the test specimens, and the positive control should be processed as the last specimen to minimize potential cross-contamination. Similarly for PCR amplification, positive (heat-inactivated culture) and negative (no template) controls should be incorporated as described for sequence capture controls. 2. Health and Safety. a. We routinely use this technique for the detection of mycobacterial species, which are Hazard Group 3 agents. For such biological agents, in the UK, Advisory Committee on Dangerous Pathogens (ACDP) guidelines (42) must be strictly adhered to, and all manipulations prior to heat inactivation at 100°C should be carried out in a safety cabinet. b. Pasteur pipets may become blocked if the specimen contains undigested tissue. Care should be taken to avoid this outcome. If blockage of the pipet occurs and attempts to release material by vigorous pipeting fail, as a last
216 Skuce et al. Fig. 3. PCR products, resulting from IS6110 IS1/IS2 amplification of NA extracted by sequence capture from lesioned tuberculous bovine lymph nodes (specimens 8, 0, 12, 19, 20) and molecular weight (M) were resolved in a 4% E-Gel (20). resort, very carefully break the glass pipet within the 20-mL specimen container. A balance must be drawn between sample loss and health and safety. c. We have found that it is not feasible to operate the FastPrep FP120 Cell Disrupter within a safety cabinet because of size limitations of the cabinet. We have also noted that it is difficult to secure the tubes in the rotor. The manufacturer’s instructions regarding securing tubes must be adhered to strictly, since tube release during operation may result in aerosol contamination of the equipment with a Category 3 pathogen. For this reason, we recommend that this equipment is not left unattended during operation and that the operator listens for evidence of tube release. The operator should respond instantly to any change in equipment tone and stop the instrument immediately. d. We consider that it is a safer practice to use a heating block rather than a boiling water bath within a safety cabinet for heat treatment at 100°C. 3. Contamination. Measures taken to minimize the risk of cross-contamination during DNA extraction and PCR amplification include the following. a. Treatment of appropriate work areas with DNA Away to eliminate contaminating DNA. b. Single use of disposable consumables including gloves, scalpels, paper plates, appropriate sized microtiter plates (serving as tube racks for screw-cap microcentrifuge and PCR tubes), and disposable ice baths.
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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.
Page 161 and 162: 164 Gallien The first description o
Page 163 and 164: 166 Gallien
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Page 167 and 168: 170 Gallien 10. Ethidium bromide: 1
Page 169 and 170: Table 2 Components (in µL) of 25-
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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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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
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Page 217 and 218: 220 Skuce et al. 14. Aranaz, A., Li
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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
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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
Page 235 and 236: 238 Hotzel et al. 3.1.5. Semen (see
Page 237 and 238: 240 Hotzel et al. Fig. 1 Detection
Page 239 and 240: 242 Hotzel et al. 4. Notes 1. The u
Page 241 and 242: 244 Hotzel et al. in Mycoplasma Pro
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Page 245 and 246: 248 Kobisch and Frey PCR assay to d
Page 247 and 248: 250 Kobisch and Frey Table 1 Oligon
Page 249 and 250: 252 Kobisch and Frey 3.2.1. Prepara
Page 251 and 252: 254 Kobisch and Frey First round of
Page 253 and 254: 256 Kobisch and Frey References 1.
Page 255 and 256: 258 Christensen et al. thrombotic m
Page 257 and 258: 260 Christensen et al. Table 1 Spec
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Page 261 and 262: 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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