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

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PCR Technology 75 negative reactions (see Chapter 2) (94). Inhibition influences the outcome of PCR by lowering the amplification efficiency or complete prevention of amplification. The inhibition acts at one or more essential stages of the reaction, the most important of which is inhibition of polymerase activity. Inhibition of PCR encountered in complex samples, such as blood, stool, serum, chicken carcass, soil, cheese, bean sprout, and oyster have been described (95,96). It has been shown that a number of substances, frequently found in enrichment media, DNA preparation solutions and food samples, inhibit PCR (97). Moreover, it was shown that different sample preparation procedures have different efficiency depending on the organism to be detected (98,99). Therefore, research in sample preparation and standardization is still needed in order to exploit the full potential of PCR in microbiological diagnosis. Not only the substances influence the outcome of the PCR, but the DNA polymerase itself can be a limiting factor because different brands of the enzyme can be inhibited by different inhibitors (46,100). Therefore, inhibition of PCR in different biological samples can be reduced or eliminated by choosing an appropriate thermostable DNA polymerase without the need for extensive sample processing prior to PCR (100). Although optimization and refinement of the PCR technique have been given much attention, the problems associated with the presence of PCR inhibitors in complex samples have not been overcome. This makes sample preparation steps prior to PCR a bottleneck in routine application and quality assurance of PCR-based methods. 6.2. Carry-over Contamination Nucleic acid amplification techniques in general, and PCR in particular, are notoriously susceptible to contamination (101,102). Once a reagent becomes contaminated with target DNA, the only solution is to discard it. It is often impossible to accurately determine which reagent is contaminated and when the contamination occurred. However, one of the greatest risks of contamination is the possibility of introducing a small amount of previously amplified DNA into a new reaction. This is referred to as “the carryover problem” (103). Enzymatic and chemical methods have been developed to control this phenomenon (104,105). One approach is based on the use of the enzyme uracil-Nglycosylase (UNG), which preferentially cleaves uracil-containing DNA. As dTTP is replaced with dUTP in the master mixture (104), UNG does not degrade native DNA templates or primers, but it cleaves the DNA products of previous amplifications at the uracil residues, rendering the contamination source nonamplifiable. UNG, which is present in the mixture from the beginning, is not active during the amplification reaction, so newly synthesized molecules are unaffected. The use of this technology is essentially invisible to the user, and no additional manipulations or equipment are necessary.
76 Lübeck and Hoorfar A simple chemical method for carryover control is based on the inactivation of amplified DNA using a photochemical technique based on isopsoralen derivatives (105). Amplification is carried out in the presence of isopsoralen. At the end, the PCR tube is irradiated with UV light, and the isopsoralens form cyclobutane monoadducts with pyrimidine bases of the DNA. If this modified DNA is carried over into subsequent amplification reactions, it will not serve as a template for amplification. However, double PCR or nested PCR cannot be controlled by either of these procedures. 7. Further Prospects 7.1. Quantitation There have been many efforts to use PCR for quantitation (106–109). There will be a further increase in the popularity of real-time PCR as commercially available equipment has become more reliable and affordable. However, great care must be taken with optimization of reaction conditions and interpretation of quantitative PCR results. Minor differences in the efficiency of amplification among samples can give rise to markedly different amounts of the final product, due to exponential nature of amplification. Ferre (110) showed, for the first time that results obtained by quantitative PCR correlated well with results from hybridization experiments. Besnard and Andre (111) developed a quantitative method in which a competitor DNA was added to the sample at known concentrations before nucleic acid extraction, so that both targets would be co-processed and quantitative results be more reproducible. The inclusion of known amounts of internal control DNA (competitors) is one of the most frequently used methodologies in quantitative PCR (75,107,109). By using this strategy, it is possible to reproducibly quantify the amount of target DNA in an environmental sample by titrating unknown amounts of target DNA against a dilution series of competitor DNA (66,75). Quantitative competitive PCR (QC-PCR) is a method consisting of co-amplification, in the same tube, of two different templates of similar length carrying the same primer binding sites. This insures both templates being amplified at equal efficiency and the ratio of products remaining constant throughout the reaction. The relative amount of each product can be determined by ethidium bromide-stained gels or by counting bands radiolabeled with 32P (112). 7.2. Automation Since the invention of thermal cyclers in connection with thermostable DNA polymerases, real-time detection equipment has introduced the second major
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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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Page 45 and 46: 46 Rådström et al. 11. Powell, H.
Page 47 and 48: 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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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.
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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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PCR Detection of Microbial Pathogens PCR Detection of Microbial ...

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