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

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STEC PCR 181 Table 7 Reaction Mix for Amplification Control Using pUC 19 DNA as Target (see Note 1) Final Concentration Component Volume (µl) in 25-µL Reaction Mix Water 11.8 — 10X buffer 2.5 1X buffer MgCl2 solution (25 mmol/L) 1.5 1.5 mmol/L dNTP-Mix (10 mmol/l) 2.0 each dNTP 200 µmol/L Primer HB 10 (20 µmol/L) 0.5 0.4 µmol/L Primer HB 11 (20 µmol/L) 0.5 0.4 µmol/L DNA polymerase 0.2 at least 1 U per mix pUC 19 DNA (1 pg/µL) 1 0.04 pg/µL Template DNA 5 — Table 8 Cleavage of PCR Amplicons by Restriction Endonucleases Virulence Factor Restriction Enzyme Fragments in bp 3. Always include a positive, a negative, and a water control. Use the strains given in Table 6 as positive controls and strain C 600 or another stx-negative strain as negative control. 4. The most important virulence factors of STEC/EHEC are stx 1, stx 2, and eae. Positive PCR results should be verified by restriction enzymes cleaving within the amplified fragment. More data are given in Table 8. In a standard procedure, the following mix should be used: 16 µL of PCR product (after running the specific temperature-time profile), 2 µL of 10X restriction buffer depending on the endonuclease (e.g., SuRE/cut buffer, Roche Diagnostics), and 2 µL (at least 20 U) of restriction enzyme. Incubate for 2 h at 37°C. After finishing, add 2 µL of gel loading buffer and start electrophoretic separation in a 2% agarose gel. Detect specific bands as described in Subheading 3.2.3. References stx 1 Acc I 91, 191 stx 2 Hae III 248, 336 eae Pst I 241, 248, 374 1. Savage, D. C. (1977) Microbial ecology of the gastrointestinal tract. Ann. Rev. Microbiol. 31, 107–133. 2. Nataro, J. P., and Kaper, J. B. (1998) Diarrheagenic Escherichia coli. Clin. Microbiol. Rev. 11, 142–201.
182 Gallien 3. Boyce, T. G., Swerdlow, D. L., and Griffin, P. M. (1995) Escherichia coli O157:H7 and the hemolytic–uremic syndrome. N. Engl. J. Med. 333, 364–368. 4. Karmali, M. A. 1989. Infection by verocytotoxin - producing Escherichia coli. Clin. Rev. 2, 15–38. 5. Karmali, M.A., Steele, B.T., Petric, M., and Lim, C. (1983) Sporadic cases of haemolytic uremic syndrome associated with faecal cytotoxin and cytotoxin- producing Escherichia coli in stools. Lancet 1, 619–620. 6. Moake, J. L. (1994) Haemolytic uraemic syndrome: basic science. Lancet 343, 393–397. 7. Remuzzi, G. and Ruggenenti, P. (1995) The hemolytic uremic syndrome. Kidney Inter. 47, 2–19. 8. Keusch, G.T. and Acheson, W.K. (1997) thrombotic thrombocytopenic Purpura associated with shiga toxins. Seminars in Hematology 34, 106–116. 9. Kaper, J. B., and O’ Brien, A. D. (ed.) (1998) Escherichia coli O157 and other Shiga toxin-producing E. coli strains. ASM Press, Washington DC, USA. 10. Karch, H., Gunzer, F., Schwarzkopf, A. and Schmidt, H. (1993) Molekularbiologie und pathogenetische Bedeutung von Shiga- und Shiga-like Toxinen. Bio Engineering 3, 39–45. 11. Riley, L. W., Remis, R. S., Helgerson, S. D., et al. (1983) Hemorrhagic colitis associated with a rare Escherichia coli serotype. New Engl. J. Med. 308, 681–685. 12. Konowalchuk, J., Speiers, J.I., and Stavrik., S. (1977) Verocell response to a cytotoxin of Escherichia coli. Infect.Immun. 18, 775–779. 13. Hovde, C. J., Calderwood, S. B., Mekalanos, J. J., and Collier, R. J. (1988) Evidence that glutamic acid 167 is an active-site residue of Shiga-like toxin 1. Proc. Natl. Acad. Sci. USA 85, 2568–2572. 14. Griffin, P. M., and Tauxe, R. V. (1991) The epidemiology of infections caused by Escherichia coli , and the associated hemolytic uremic syndrome. Epidemiol. Rev. 13, 60–98. 15. Scotland, S. M., Willshaw, G. A., Smith, H. R., and Rowe, B. (1990) Properties of strains of Escherichia coli O26:H11 in relation to their enteropathogenic or enterohemorrhagic classification. J. Infect. Dis. 162, 1069–1074. 16. Scheutz, F. (1995) Epidemiology of non-O 157 VTEC. Proceedings of WHO Consultation on Emerging Food-borne Diseases, Berlin , March 20–24, 1995. 17. Tarr, P. I., and Neill, M. A. (1996) Perspective: the problem of non-O157 : H7 shiga toxin (verocytotoxin) - producing Escherichai coli. J. Infect. Dis. 174, 1136–1139. 18. Willshaw, G. A., Smith, H. R., Roberts, D., Thirlwell, J., Cheasty, T., and Rowe, B. (1993) Examination of raw beef products for the presence of vero cytotoxin producing Escherichia coli , particularly those of serogroup O157. J. Appl. Bacteriol. 75, 420–426. 19. Datz, M., Janetzki-Mittmann, C., Franke, S., Gunzer, F., Schmidt, H., and Karch, H. (1996) Analysis of the enterohemorrhagic Escherichia coli O157 DNA region containing lambdoid phage gene p and shiga - like - toxin structural genes. Appl. Environml. Microbiol. 62, 791–797.
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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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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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Page 141 and 142: 144 Popoff Taq reaction buffer 1X,
Page 143 and 144: 146 Popoff almost all C. perfringen
Page 145 and 146: 148 Popoff 2. Add to each PCR tube
Page 147 and 148: 150 Popoff 7. Hielm, S., Hyyttia, E
Page 149 and 150: 152 Popoff 35. Wieckowski, E., Bill
Page 151 and 152: 154 Berri et al. 2. Materials 1. C.
Page 153 and 154: 156 Berri et al. 3.3.2. Vaginal Swa
Page 155 and 156: 158 Berri et al. Fig. 1. Trans-PCR.
Page 157 and 158: 160 Berri et al. Fig. 3. Sensitivit
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Page 161 and 162: 164 Gallien The first description o
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Page 183 and 184: 186 O'Connor This chapter will desc
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Page 199 and 200: 202 Skuce et al. Table 1 Significan
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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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264 Christensen et al. 3.2. Extract
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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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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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284 Malorny and Helmuth Table 4 PCR
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292 Wastling and Mattsson 2. Remove
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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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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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