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

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Pre-PCR Processing 37 plicity and low cost. The method provides viable cells to be used in PCR without further lysis steps (34). However, it must be borne in mind that cells contain high concentrations of macromolecules, which might influence and shift the equilibrium in many biochemical reactions (35), for instance the DNA polymerase and its DNA template–primer binding properties (36) (see Chapter 1). Therefore, the DNA polymerase has a key function during DNA amplification in terms of DNA synthesis and resistance to PCR inhibitors. A comparison of the performance of sample preparation methods described in this section is shown in Table 2. 4. DNA Polymerases The first PCR experiments were carried out with the thermolabile Klenow fragment of Eschericia coli DNA polymerase I, which needed to be replenished for every cycle (37). The use of the thermostable DNA polymerase from Thermus aquaticus (Taq) has greatly simplified PCR and enhanced the specificity (38). With high specific activity, fidelity, and temperature range, Taq DNA polymerase and its derivatives became and still are the most widely used enzymes in PCR. Thermostable DNA polymerase is a key component in the amplification reaction, and any factor interfering with the enzymatic activity will affect the amplification capacity. The DNA polymerase can be degraded, denatured, or have its enzymatic activity reduced by a wide variety of compounds present in biological samples (3,5,9,39). A number of DNA polymerases from other organisms are now commercially available. Examples of commonly used DNA polymerases include rTth and Tth, isolated from Thermus thermophilus, DyNazyme isolated from T. brockianus, as well as AmpliTaq ® Gold (Applied BioSystems, Foster City, CA, USA) and Platinum Taq with built-in hot start, both isolated from T. aquaticus. These polymerases exhibit very different properties with regard to resistance to various components in biological samples and performance in the presence of these components. The choice of DNA polymerase was shown to influence the performance of several PCR-based applications, such as genotyping using restriction fragment-length polymorphism (RFLP) (40) and random-amplified polymorphic DNA (RAPD) (41), multiplex PCR assays (42), differential display reverse transcription PCR (RT-PCR) (43), and autosticky PCR (44). Recent research indicated that different polymerases have different susceptibilities to PCR inhibitors (2). Therefore, the inhibition of PCR by components of biological samples can be reduced or eliminated by choosing an appropriate thermostable DNA polymerase without the need for extensive sample processing prior to PCR. The choice of DNA polymerase is determined by several factors related to the application. The level of resistance of DNA polymerase to PCR inhibitors
Table 2 Comparison of the Performance of Different Pre-PCR Sample Preparation Methods Product Category of sample of sample Homogeneity Concentration Removal of Time preparation method preparation of product of product PCR inhibitors required Cost Availability Biochemical: DNA extraction DNA Good Average Yes 3–6 h High Complex Immunological: Immunomagnetic capture Cell/DNA Average Average Average 2–4 h High Limited Physical: Buoyant density centrifugation Cell Average Good Average 30 min Average Limited Physiological: Enrichment Cell Low Good Low 6–24 h Low Good 38 Rådström et al.
Page 1 and 2: TM Methods in Molecular Biology VOL
Page 3 and 4: 4 Sachse 2. Selection of Target Seq
Page 5 and 6: Table 1 Target Sequences Used in PC
Page 7 and 8: 8 Sachse Fig. 1. Structural organiz
Page 9 and 10: 10 Sachse proteins (66-68), invasio
Page 11 and 12: 12 Sachse Since there appears to be
Page 13 and 14: 14 Sachse In the context of identif
Page 15 and 16: 16 Sachse to partially compensate t
Page 17 and 18: 18 Sachse nostic potential of PCR.
Page 19 and 20: 20 Sachse product has to be confirm
Page 21 and 22: 22 Sachse 7. van Camp, G., Fierens,
Page 23 and 24: 24 Sachse 33. Rappelli, P., Are, R.
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Page 27 and 28: 28 Sachse 89. Bloch, W. (1991) A bi
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Page 33 and 34: 34 Rådström et al. immunoglobulin
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Page 39 and 40: 40 Rådström et al. explain these
Page 41 and 42: 42 Rådström et al. 5.1. Proteins
Page 43 and 44: 44 Rådström et al. neous. Consequ
Page 45 and 46: 46 Rådström et al. 11. Powell, H.
Page 47 and 48: 48 Rådström et al. 39. Katcher, H
Page 49 and 50: 50 Rådström et al. 73. Nagai, M.,
Page 51 and 52: 52 Hoorfar and Cook 2. FOOD-PCR: A
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Page 65 and 66: 66 Lübeck and Hoorfar ods for the
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Page 85 and 86: 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.
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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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