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

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Pre-PCR Processing 35 Table 1 Sample Preparation Methods Used for Different Types of Samples a Category of sample preparation Sample method Subcategory preparation method Sample Reference Biochemical Adsorption Lectin-based separation Beef meat (78) Protein adsorption Blood (9) DNA extraction DNA purification method Hemolytic serum (79) Lytic methods Blood anticoagulant (80) Immunological Adsorption Immunomagnetic capture Blood (81) Physical Aqueous two-phase systems Soft cheese (82) Buoyant density centrifugation Minced meat (31) Centrifugation Urine (28) Dilution Blood (30) Filtration Milk (29) Physiological Enrichment Meat (33) a Modified with permission from Ref. 1. 3.1. Biochemical Methods The most widely employed biochemical method is DNA extraction. Many different commercial kits are available, such as BAX (Quallcon Inc., Wilmington, DE) (16), PrepMan (Applied BioSystems, Foster City, CA, USA) (17), Purugene (Gentra Systems Inc., Minneapolis, MN, USA) (18), QIAamp ® (Qiagen, Valencia, CA, USA) (19), and XTRAX (Gull Laboratories Inc., Salt Lake City, UT, USA) (20). Consequently, several studies have compared and evaluated the quality of the extracted DNA (18,21,22), and a kit that provides the highest yield, concentration, and purity of DNA can be recommended. The advantage of DNA extraction is that a homogeneous sample with high quality is provided for amplification. Most PCR inhibitors are removed, since the template is usually purified and stored in appropriate buffers, such as Tris-EDTA (TE) buffer. The drawback of DNA extraction methods is that the target microorganism usually has to be pre-enriched in medium or on an agar plate prior to extraction. In addition, most DNA extraction methods are laborious and costly. Batch-to-batch variation after DNA extraction may also exist with respect to purity and concentration of the template. 3.2. Immunological Methods This category is mainly based on the use of magnetic beads coated with antibodies (23). Since antibodies are used, the specificity will be influenced,
36 Rådström et al. and the captured cells will be those containing the corresponding antigen. The specificity of the PCR protocol will depend on both the PCR assay used, as well as the specificity of the antibodies. In general, after immunocapture, the sample requires lysis or washing (24), and viruses can then be used directly (25). In most cases, these methods increase the concentration of the target organism. The homogeneity of the PCR sample may differ depending on the processing steps that follow the capture, but usually the template is of appropriate quality after this treatment. Since part of the specificity depends on the antibodies themselves, false negative results can be obtained as a result of cross-reactions. This methodology is quite expensive and also very laborious and time-consuming. 3.3. Physical Methods Many different physical methods have been used, such as aqueous two-phase systems (26), buoyant-density centrifugation (27), centrifugation (28), filtration (29) and dilution (30). These methods are dependent on the physical properties of the target cells, for example cell density and size. Aqueous two-phase systems provide a gentle way of partitioning PCR inhibitors and target cells between two immiscible phases. For instance, a polyethylene glycol (PEG) 4000 and dextran 40-based system was used in a PCR detection assay for Helicobacter pylori in human feces (6). Density centrifugation was shown to be a promising method if fast detection is of importance (31). Density media, such as Percoll (Pharmacia, Uppsala, Sweden) (27) and BactXtractor (Quintessence Research AB, Bålsta, Sweden) (32), were used to concentrate the target organism and remove PCR-inhibitory substances of different density. After this treatment, whole cells were obtained, which could be used as a PCR sample. The homogeneity of the sample may differ depending on the kind of biological sample matrices. If components of the sample matrix have the same density as the cells these may inhibit DNA amplification. The advantage of density centrifugation is that the target organism is being concentrated, which allows rapid detection response. Furthermore, these methods are relatively user friendly. 3.4. Physiological Methods These methods are based on bacterial growth and biosynthesis of cell components, i.e., genome, cytoplasm, and cell surface constituents. Culture can be carried out in enrichment broth or on agar plates. Again, the aim is to provide detectable concentrations of viable target cells prior to PCR (33). Selective or nonselective agar or enrichment medium can be used, and the specificity will depend partly on the characteristics of the medium. The template quality, as well as the homogeneity of the PCR sample, may differ with respect to the presence of cell components. The advantages of this methodology are its sim-
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
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Page 17 and 18: 18 Sachse nostic potential of PCR.
Page 19 and 20: 20 Sachse product has to be confirm
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Page 23 and 24: 24 Sachse 33. Rappelli, P., Are, R.
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Page 51 and 52: 52 Hoorfar and Cook 2. FOOD-PCR: A
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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.
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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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