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

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Identification of B. abortus by PCR 103 Fig. 1. Predicted amplified loci (rows) for various categories of unknowns (columns). (A) The four loci for each category are shown with their hybridizing primers; (B) the predicted products resulting from successful amplification. , 16S locus; , alkB locus; , IS711; , wboA locus; , eri locus; , DNA absent; , hybridizing amplification primer; , nonhybridizing primer. A 500-bp product from the IS711-alkB locus is amplified with all B. abortus (biovar 1, 2, and 4) templates including those from the vaccine strains. Other Brucella species and bacteria do not have this DNA sequence, and no product will be amplified. A 300-bp product will be amplified from the IS711-wboA locus found only in the vaccine strain RB51. A 180-bp product is amplified from the eri gene present in all Brucella species and strains except B. abortus vaccine strain S19. Other bacterial genomes lack this locus and will not amplify the 180-bp product. Sample results are shown in Fig. 2. 3.6. Troubleshooting The B. abortus RB51 positive control should amplify all four products. If some or all of the products are missing, then there was a problem with the Master Mixture or the cycling parameters. Repeat the assay with fresh Master Mixture. The presence of amplification in the negative controls indicates a contamination problem. If both negative controls are contaminated, then the entire batch of Master Mixture was probably contaminated and should be discarded.
104 Ewalt and Bricker Fig. 2. Typical patterns amplified from bacterial bovine isolates as detected by agarose gel electrophoresis. Lane 1, 100-bp ladder; lane 2, B. abortus RB51; lane 3, B. abortus field strain; lane 4, B. abortus field strain; lane 5, B. abortus field strain; lane 6, B. abortus field strain; lane 7, B. abortus field strain; lane 8, B. abortus RB51; lane 9, B. abortus field strain; lane 10, B. abortus field strain; lane 11, B. abortus strain S19; lane 12, Brucella species (not B. abortus); lane 13, Brucella species (not abortus); and lane 14, non-Brucella bacteria. A 2% agarose gel was loaded with 8-µL amplified product and 1-µL loading dye per well, electrophoresed for 2.5 h at 70 V, stained with ethidium bromide, and visualized with UV light. If only the water control is contaminated, then it means the problem probably occurred during the current assay, and the rest of the unused batch is probably usable. Repeat the assay with fresh water and extra care to prevent contamination. If the contamination of either control amplifies only the large 16S gene product, then any exposure to bacteria during the process could have caused the contamination. This may be difficult to avoid since even commercial enzymes and buffers may contain trace levels of nonspecific bacterial DNA, and absolute sterility in the larger laboratory environment is difficult to maintain. The appearance of weak bands on a gel can complicate the analysis. Weak bands can result from cross-well contamination, improper amplification parameters or sample contamination. Only a few bacteria contaminating an isolate can result in visible amplified product. Multiplex PCR is more sensitive to variations in the amplification parameters than is traditional PCR. The annealing temperature is a good example. The addition of more primers increases the range of individual optimal melting temperatures (T ms) and also the potential for mispriming events. The optimal temperature for each primer is a compromise between a lower temperature for stable annealing to the correct locus (but also
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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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Page 51 and 52: 52 Hoorfar and Cook 2. FOOD-PCR: A
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Page 85 and 86: 88 Frey Table 1 Presence of the Var
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Page 135 and 136: 138 Popoff spastic paralysis. The g
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Page 141 and 142: 144 Popoff Taq reaction buffer 1X,
Page 143 and 144: 146 Popoff almost all C. perfringen
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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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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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