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

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Toxoplasma PCR 291 2. 50X TAE buffer: 242g Tris base, 57.1 mL glacial acetic acid, and 100 mL 0.5 M EDTA, pH 8.0 per L, final concentration 2 M Tris-acetate, 50 mM EDTA. 3. 10X TBE buffer: 108 g Tris base, 15.5 mL 85% phosphoric acid, and 40 mL 0.5 M EDTA, pH 8.0, per L, final concentration 0.45 M Tris-borate, 10 mM EDTA. 4. 30% Acrylamide/bis solution, 19:1; TEMED, and 10% Ammonium persulphate solution (Bio-Rad, Hercules, CA, USA). 5. Ethidium bromide, 10 mg/mL (Bio-Rad). Note: Wear gloves when handling. 6. Gel loading solution (GLS) (6X): 0.25% bromophenol blue, 0.25% xylene cyanol FF, 40% (w/v) sucrose in water. 7. Molecular weight marker: 50-bp ladder (Amersham Biosciences). 8. Agarose and/or polyacrylamide gel electrophoresis equipment. 9. UV-transilluminator. 2.4. Southern Blotting 1. Transfer solution: 3 M NaCl and 8 mM NaOH. 2. Positively charged Nylon membranes (Roche Molecular Biochemicals, Mannheim, Germany). 3. 20X SSC: 3 M NaCl, 0.3 M sodium citrate, pH 7.0. 4. PCR DIG Probe synthesis kit (Roche Molecular Biochemicals). 5. GENECLEAN ® (Qbiogene, San Diego, CA,USA). 6. DIG Easy Hyb solution (Roche Molecular Biochemicals). 7. Wash solution, containing (2X SSC, 0.1% sodium dodecyl sulfate (SDS). 8. Wash solution (0.5X SSC 0.1% SDS). 9. DIG Nucleic Acid Detection Kit (Roche Molecular Biochemicals). 3. Methods 3.1. Sample Preparation The methods described below outline sample preparation for (i) whole blood, (ii) lymph, and (iii) tissue samples. Note that vol and quantities of starting material can be scaled-up or -down according to sample type and availability (see Note 1). 3.1.1. Whole Blood T. gondii is an intracellular parasite that can invade virtually any nucleated cell, but is unable to survive in RBCs. To detect the parasite in blood samples, it is usually best to isolate the white cells, removing as many RBCs as possible since their contents can be inhibitory to the PCR. 1. Dilute the fresh blood sample (5–10 mL) 50:50 with PBS and spin at 1000g for 10 min. This will partition the cells, so that an interface of white cells forms between the RBCs at the bottom of the tube and a straw-colored plasma layer at the top of the tube.
292 Wastling and Mattsson 2. Remove the tube from the centrifuge and using a plastic pipet, carefully remove the interface containing the white cells. Mix this with 2 mL PBS. 3. Layer the diluted white cells over Lymphoprep (10 mL) and spin at 1000g for 15 min to clean the cells further. Again, carefully remove the white cell interface using a plastic disposable pipet and decant it into a clean tube. 4. At this stage, some RBCs may remain, and these are best removed by lysis with ammonium chloride. To do this, treat the sample with an equal volume of 10 mM Tris-NH 4Cl for 2 min at room temperature and then wash the resulting cell suspension 3× by resuspension in PBS and centrifugation at 1000g for 10 min. The pellet may now be stored frozen at –20°C for later processing if desired. 5. Resuspend the resulting pellet in 50 µL of 10 mM Tris, pH 8.3, 40 mM KCl, 2.5 mM MgCl 2 containing proteinase K 100 µg/mL and Tween 20 (0.5%). 6. Incubate the mixture at 55°C for 1 h and then inactivate the proteinase K by boiling for 5 min. 7. Store the samples at –20°C for later PCR analysis (see Note 2). 3.1.2. Lymph 1. Dilute the lymph sample 1 in 10 with PBS to prevent clotting and centrifuge at 1000g for 10 min. Resuspend cells in 1 mL PBS. 2. Lyse any contaminating RBCs by the addition of 1 mL of 10 mM Tris-NH4Cl and wash the remaining cells 3× by resuspension in PBS and centrifugation at 1000g for 10 min. The pellet may now be stored frozen at -20°C for later processing. 3. Resuspend the resulting pellet in 50 µL of 10 mM Tris, pH 8.3, 40 mM KCl, 2.5 mM MgCl 2 containing proteinase K 100 µg/mL and Tween 20 (0.5%). 4. Incubate the mixture at 55ºC for 1 h and then inactivate the proteinase K by boiling for 5 min. 5. Store the samples at -20°C for later PCR analysis (see Note 3). 3.1.3. Tissue Samples PCR can be performed either on fresh or frozen tissue. However, if tissue is to be frozen before analysis care should be taken to freeze samples at –20°C as soon as possible after collection as autolysis of the sample and degradation of DNA may occur quite rapidly. 1. For processing for PCR, finely chop the samples and wash with ice-cold PBS. 2. Digest the tissues overnight at 37°C in 100 µL of 50 mM Tris, pH 8.5, 1 mM EDTA, Tween 20 (0.5%) containing proteinase K 200 µg/mL. 3. After digestion, inactivate the proteinase K by boiling for 5 min. 4. Store the samples at –20°C for later PCR analysis. 3.2. DNA Amplification Detection of the parasite is by amplification of the T. gondii repetitive B1 locus.
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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.
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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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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
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Page 253 and 254: 256 Kobisch and Frey References 1.
Page 255 and 256: 258 Christensen et al. thrombotic m
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Page 267 and 268: 270 Christensen et al. 7. To reduce
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Page 271 and 272: 274 Christensen et al. 54. Hunt, M.
Page 273 and 274: 276 Malorny and Helmuth valent meta
Page 275 and 276: 278 Malorny and Helmuth 12. Apparat
Page 277 and 278: 280 Malorny and Helmuth 3.3. Amplif
Page 279 and 280: 282 Malorny and Helmuth Table 2 Vol
Page 281 and 282: 284 Malorny and Helmuth Table 4 PCR
Page 283 and 284: 286 Malorny and Helmuth 3. Wallace,
Page 285 and 286: 288 Malorny and Helmuth
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Page 291 and 292: 294 Wastling and Mattsson Fig. 1. B
Page 293 and 294: 296 Wastling and Mattsson time PCR
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Page 297 and 298: 300 Pozio and La Rosa Table 1 Princ
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Page 301 and 302: 304 Pozio and La Rosa 3. Proteinase
Page 303 and 304: 306 Pozio and La Rosa Table 3 PCR-R
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Page 309 and 310: 312 Knutsson and Rådström The ref
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Page 313 and 314: 316 Knutsson and Rådström 2.4. El
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Page 319 and 320: 322 Knutsson and Rådström 4. Alek
Page 321: 324 Knutsson and Rådström 34. Hee
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PCR Detection of Microbial Pathogens PCR Detection of Microbial ...

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