PCR for Clinical Microbiology (eBook)

Foreword 5
Contents 7
Contributors 13
Abbreviations 19
Introduction 25
References 27
Part I PCR Fundamentals 28
1 PCR Basics 29
1.1 Introduction 29
1.2 Facility Layout, Airflow and Workflow 31
1.3 Good Molecular Biology Laboratory Practices 34
References 36
2 PCR Methodology 37
2.1 Nucleic Acid Extraction, Purification and Storage 37
2.2 Conventional PCR 45
2.2.1 Introduction 45
2.2.2 Components of PCR 45
2.2.3 PCR Amplification and Product Detection 46
2.2.4 Conventional PCR in the Diagnostic Laboratory 46
2.2.5 Limitations of Conventional PCR 47
2.2.6 Summary 47
2.3 Real-Time PCR 48
2.3.1 Introduction 48
2.3.2 Real-Time PCR Technology 48
2.3.3 Real-Time PCR in the Diagnostic Laboratory 51
2.3.4 Instrumentation for Real-Time PCR 51
2.3.5 Considerations in the Use of Real-Time PCR 52
2.3.6 Summary 53
2.4 Quantitative PCR 53
2.4.1 Introduction 53
2.4.2 PCR Kinetics and Q-PCR 54
2.4.3 Absolute Quantification with External Standards 55
2.4.4 Absolute Quantification Using Internal Controls 56
2.4.5 Detection Formats 56
2.4.5.1 Intercalating Dyes 56
2.4.5.2 Sequence-Specific Fluorescent Dyes 56
2.4.6 Clinical Use 57
2.4.7 Conclusion 57
2.5 Multiplex PCR in Diagnostic Microbiology 57
2.5.1 Introduction 58
2.5.2 Multiplex Technologies 59
2.5.3 Practical Applications 59
2.6 Molecular Subtyping 60
2.6.1 Introduction 60
2.6.2 Why Bacterial Typing Is Performed 61
2.6.3 Typing Methods Based on Phenotype 62
2.6.4 Typing Methods Based on Electrophoresis of DNA Fragments Derived from the Whole Genome 63
2.6.5 Typing Methods Based upon Known Polymorphic Genes or Sites 64
2.6.6 Projections of Future Developments 68
References 68
3 Laboratory Accreditation Standards 74
3.1 Quality in the Medical Molecular Microbiology Laboratory 74
3.1.1 Part 1: Overview 74
3.1.1.1 Quality and the Role of the Laboratory 74
3.1.1.2 Legislating for Quality 75
3.1.1.3 Quality and Standards 75
3.1.1.4 Quality and Regulation 76
3.1.2 Part 2: Specific Aspects Relating to Quality 77
3.1.2.1 Risk Management 77
3.1.2.2 Validation and Verification 78
3.1.2.3 Quality Assurance 78
3.1.2.4 Conclusion 79
3.1.3 Annex 79
3.1.3.1 Compendium of Standards and Guidelines for Medical Molecular Microbiological Laboratories 79
3.1.3.2 European Committee for Standardization 80
3.1.3.3 Clinical and Laboratory Standards Institute (CLSI) Standards 81
3.2 Security Sensitive Biological Agents (SSBA) and Office of the Gene Technology Regulator (OGTR) Standards 82
3.2.1 Security Sensitive Biological Agents (SSBA) Regulatory Scheme 82
3.2.2 Regulation of Gene Technology 83
Part II Clinical Aspects and Diagnosis of Infectious Disease 85
4 Meningitis and Encephalitis 86
References 89
5 Respiratory Infections 90
5.1 Introduction 90
5.2 Clinical Aspects and Epidemiology 92
5.2.1 Clinical Presentation 92
5.2.2 Pathogenesis (Transmission, Incubation, Site of Infection) 92
5.2.3 Epidemiology (Frequency, Seasonality, Age Groups) 93
5.3 Commonly Recognised Respiratory Agents 93
5.3.1 Respiratory Disease Due to Viruses 93
5.3.2 Respiratory Tract Infections Due to Bacteria 95
5.3.3 Respiratory Infections Due to Fungi 96
5.4 New Viruses Associated with the Respiratory Tract 97
5.4.1 Human Metapneumovirus 98
5.4.2 SARS Coronavirus 99
5.4.3 Human Coronaviruses NL63 and HKU1 99
5.4.4 Human Bocavirus 100
5.4.5 Novel Human Polyomaviruses KIV, WUV and MCV 100
5.5 Laboratory Diagnosis 101
5.5.1 Respiratory Specimens and Transport 101
5.5.2 Traditional Methods 102
5.5.3 Criteria for Test Selection 102
5.5.4 Commercial Assays 103
5.6 PCR Detection of Respiratory Viruses 103
References 104
6 Blood-Borne Disease 106
6.1 PCR Use in the Diagnosis of Blood-Borne Viral Diseases 107
6.1.1 Introduction 107
6.1.2 Qualitative NAT 107
6.1.3 NAT in the Blood Supply 108
6.1.4 Quantitative NAT 108
6.1.5 Genotyping 111
6.1.6 Molecular Epidemiology 111
6.2 PCR Use in the Diagnosis of Infection in the Immunocompromised Host 113
6.2.1 Background 113
6.2.2 Issues to Consider in the Diagnosis of Infection in the Immunocompromised Host 114
7 Enteric Infections 117
7.1 Introduction 117
References 119
8 Sexually Transmitted Infections 120
8.1 Introduction to Sexually Transmitted Infections 120
8.2 Clinical Aspects and Epidemiology 121
8.2.1 Clinical Presentation 121
8.2.2 Genital HSV 122
8.2.3 Chlamydia trachomatis 122
8.3 Commonly Recognised STI Agents 127
8.4 Laboratory Diagnosis 131
8.4.1 Specimens and Transport 131
8.4.2 Traditional Methods 132
8.4.3 Criteria for Test Selection 132
8.4.4 Commercial Assays 132
8.5 PCR Diagnosis of Sexually Transmitted Infections 133
8.5.1 Technical Considerations 133
8.5.2 Which Technology, Platform, Reaction Mix? 134
8.5.3 Quality Control Issues 134
References 135
Part III PCR Protocols for Bacterial Pathogens 137
9 Bordetella pertussis and Bordetella parapertussis 138
9.1 Acceptable Specimens 138
9.2 Sample Extraction 138
9.3 Primers and Probe Sequences 138
9.4 PCR Amplification and Product Detection 139
9.5 PCR Amplification and Product Detection 140
9.5.1 Quality Control and Validation Data 140
9.6 Assay Limitations 142
References 142
10 Brucellosis The Role of PCR in Diagnosis and Management 143
10.1 Clinical Implications 143
10.2 Assay Descriptions 144
10.2.1 Brucella spp. LightCycler Real-Time PCR 144
10.2.2 Gel Based PCR Method 144
10.3 Safety 145
10.4 Acceptable Specimens 145
10.5 Extraction Procedure 146
10.6 Primer and Probe Sequences 146
10.6.1 Brucella spp. LightCycler Real Time PCR 146
10.6.2 Primer/Probe Sequences: Brucella spp. Gel Based PCR 146
10.7 PCR Amplification Conditions and Product Detection 146
10.7.1 Brucella spp. LightCycler Real Time PCR 147
10.7.2 Brucella spp. Gel Based PCR 147
10.8 Quality Control 149
10.9 Validation Data 149
References 149
11 Burkholderia cepacia Complex and Burkholderia gladioli 150
11.1 Summary of Methods 150
11.2 Background 150
11.3 Acceptable Specimens 151
11.4 Sample Extraction 151
11.5 Primer Sequences 151
11.6 PCR Amplification and Product Detection 151
11.7 Quality Control and Validation Data 153
11.8 Assay Limitations 153
References 153
12 Chlamydia and Lymphogranuloma Venereum (LGV) 154
12.1 Summary of Methods 154
12.2 Acceptable Specimens 154
12.3 Unacceptable Specimens 154
12.4 Sample Extraction 155
12.5 Primers and Probe Sequences 155
12.6 PCR Amplification and Product Detection 155
12.7 Quality Control and Validation Data 156
12.8 Assay Limitations 156
References 156
13 Chlamydophila 157
13.1 Summary of Methods 157
13.2 Clinical Background 157
13.3 Acceptable Specimens 158
13.4 Unacceptable Specimens 158
13.5 Sample Extraction 159
13.6 Primers and Probe Sequences 159
13.7 PCR Amplification and Product Detection 159
13.8 Quality Control and Validation Data 160
13.9 Assay Limitations 160
References 160
14 Coxiella burnetii 161
14.1 Introduction 161
14.2 Assay Description 162
14.3 Acceptable Specimens 162
14.4 Sample Extraction 162
14.5 Primer and Probe Sequences 163
14.6 PCR Amplification and Analysis 163
15 Diarrheagenic Escherichia coli Pathotypes (DEP) Including Enterohaemorrhagic (EHEC)/Shiga-toxin E. coli (STEC) 165
15.1 Summary of Methods 165
15.2 Organism 166
15.3 Characterisation of DEPs 166
15.4 Sample Extraction 166
15.5 PCR Amplification and Product Detection 168
15.6 Identification of DEPs 168
15.7 O-Molecular (OMO) Typing of DEPs 169
15.8 Conclusion 171
References 171
16 Haemophilus ducreyi and Klebsiella granulomatis 172
16.1 Summary of Methods 172
16.2 Acceptable Specimens 172
16.3 Sample Extraction 172
16.4 Primers and Probe Sequences 173
16.5 PCR Amplification and Product Detection 173
16.5.1 Multiplex PCR Screen 173
16.5.2 K. granulomatis Confirmation 173
16.6 Quality Control and Validation Data 175
16.7 Assay Limitations 175
References 175
17 Haemophilus influenzae Capsulated and Non-typeable 176
17.1 Summary of Methods 176
17.2 Background 176
17.3 Acceptable Specimens 177
17.4 Sample Extraction 178
17.5 Primers and Probe Sequences 178
17.6 PCR Amplification and Product Detection 178
17.7 Quality Control and Validation Data 178
References 179
18 Mycobacterium tuberculosis Complex 180
18.1 Summary of Methods 180
18.2 Acceptable Specimens 180
18.3 Sample Extraction 181
18.4 Primers and Probe Sequences 182
18.5 Result Interpretation and Reporting 183
19 Mycobacteria Other Than Mycobacterium tuberculosis 186
19.1 Summary of Methods 186
19.2 Introduction 186
19.3 Acceptable Specimens 187
19.4 Unacceptable Specimens 187
19.5 Sample Extraction 187
19.6 Primer Sequences 187
19.7 First PCR Amplification (Mb246 + MbR247) 188
19.8 Nested PCR Amplification (Mb1 + MbR7) 188
19.9 Product Detection 188
19.10 Pre-sequencing Treatment 189
19.11 DNA Sequencing 189
19.12 DNA Sequence Analysis 189
19.13 Reporting 189
19.14 Quality Control 189
19.15 Assay Limitations 190
References 190
20 Mycoplasma genitalium 191
20.1 Summary of Methods 191
20.2 Acceptable Specimens 191
20.3 Sample Extraction 191
20.4 Primers and Probe Sequences 191
20.5 PCR Amplification and Product Detection 192
20.6 Quality Control and Validation Data 192
20.7 Assay Limitations 192
References 192
21 Mycoplasma pneumoniae 193
21.1 Summary of Methods 193
21.2 Background 193
21.3 Acceptable Specimens 194
21.4 Unacceptable Specimens 194
21.5 Sample Extraction 194
21.6 Primer and Probe Sequences 194
21.7 PCR Amplification and Product Detection 195
21.8 Quality Control and Validation Data 195
21.9 Assay Limitations 196
References 196
22 Neisseria gonorrhoeae 197
22.1 Summary of Methods 197
22.2 Acceptable Specimens 197
22.3 Unacceptable Specimens 197
22.4 Sample Extraction 197
22.5 Primers and Probe Sequences 198
22.6 PCR Amplification and Product Detection 198
22.7 Quality Control and Validation Data 198
22.8 Assay Limitations 199
References 199
23 Neisseria meningitidis 200
23.1 Summary of Methods 200
23.2 Organism 200
23.3 Acceptable Specimens 201
23.4 Unacceptable Specimens 201
23.5 Sample Extraction 201
23.6 Primer and Probe Sequences 201
23.7 PCR Amplification and Product Detection 202
23.8 Quality Control and Validation Data 202
23.9 Assay Limitations 202
23.10 Serogroup Determination 202
References 203
24 Pseudomonas aeruginosa 204
24.1 Summary of Methods 204
24.2 Background 204
24.3 Acceptable Specimens 205
24.4 Unacceptable Specimens 205
24.5 Sample Extraction 205
24.6 Primers and Probe Sequences 205
24.7 PCR Amplification and Product Detection 206
24.8 Quality Control and Validation Data 206
24.9 Assay Limitations 207
References 207
25 Rickettsia 209
25.1 Introduction 209
25.2 Assay Description 209
25.3 Acceptable Specimens 210
25.4 Unacceptable Specimens 210
25.5 Sample Extraction 210
25.6 Primer and Probe Sequences 211
25.7 PCR Amplification and Analysis 211
Reference 211
26 Streptococcus pneumoniae 212
26.1 Summary of Methods 212
26.2 Acceptable Specimens 212
26.3 Unacceptable Specimens 212
26.4 Sample Extraction 213
26.5 Primer and Probe Sequences 213
26.6 PCR Amplification and Product Detection 213
26.7 Quality Control and Validation Data 213
26.8 Assay Limitations 214
References 214
27 Treponema pallidum 215
27.1 Summary of Methods 215
27.2 Acceptable Specimens 215
27.3 Sample Extraction 216
27.4 Primers and Probe Sequences 216
27.5 PCR Amplification and Product Detection 216
27.6 Quality Control and Validation Data 217
27.7 Assay Limitations 217
References 218
28 Universal Bacterial Identification by PCR and DNA Sequencing of 16S rRNA Gene 219
28.1 Summary of Method 219
28.2 Introduction 219
28.3 Acceptable Specimens 220
28.4 Unacceptable Specimens 220
28.5 Sample Extraction 220
28.6 Primer Sequences 220
28.7 PCR Amplification 221
28.8 Product Detection 222
28.9 Pre-sequencing Treatment 222
28.10 DNA Sequencing 222
28.11 DNA Sequence Analysis 222
28.12 Reporting 223
28.13 Quality Control 223
28.14 Assay Limitations 224
References 224
Part IV PCR Protocols for Viral Pathogens 225
29 Adenovirus 226
29.1 Summary of Methods 226
29.2 Organism 226
29.3 Sample Preparation and DNA Extraction 227
29.4 Primer Sequences 227
29.5 PCR Amplification and Product Detection 227
References 228
30 Cytomegalovirus (CMV) 229
30.1 CMV gp58 PCR 229
30.1.1 First Round Amplification 229
30.1.2 Second Round Amplification 230
30.2 CMV MIE PCR 230
30.2.1 First Round Amplification 230
30.2.2 Second Round Amplification 231
30.3 Primer Sequences 231
30.4 Sample Extraction 232
30.5 Detection of PCR Products 232
References 232
31 Dengue Virus 233
31.1 Summary of Methods 233
31.2 Organism 233
31.3 Acceptable Specimens 234
31.4 Sample Extraction 234
31.5 Primer and Probe Sequences 234
31.6 Amplification Conditions 235
31.7 Quality Control and Validation Data 235
31.8 Assay Limitations and Alternative Protocols 236
References 236
32 Enterovirus 237
32.1 Summary of Methods 237
32.2 Acceptable Specimens 238
32.3 Unacceptable Specimens 238
32.4 Sample Extraction 238
32.5 Primer and Probe Sequences 239
32.6 PCR Amplification and Product Detection 239
32.6.1 Semi-nested Endpoint PCR 239
32.6.2 TaqMan RT-qPCR 239
32.7 Quality Control and Validation Data 240
References 241
33 Epstein Barr Virus 242
33.1 Summary of Methods 242
33.2 Introduction 242
33.3 Specimen Collection 243
33.4 Specimen Preparation 243
33.5 Primer and Probe Sequences 244
33.6 Equipment 244
33.7 PCR Amplification and Product Detection 244
33.8 EIA Detection of PCR Products 245
33.9 Hybridisation Solution 245
33.10 Conjugate 246
33.11 Quality Control 246
Reference 247
34 Flavivirus 248
34.1 Summary of Methods 248
34.2 Acceptable Specimens 248
34.3 Sample Extraction 249
34.4 Primer and Probe Sequences 249
34.5 PCR Amplification and Product Detection 249
34.6 Quality Control and Validation Data 251
34.7 Assay Limitations 251
References 251
35 Hepatitis A Virus 252
35.1 Clinical Background 252
35.2 Acceptable Specimens 253
35.3 Unacceptable Specimens 253
35.4 Sample Extraction 253
35.5 Primer Sequences 253
35.6 PCR Amplification and Product Detection 254
35.7 Quality Control and Validation Data 254
Reference 255
36 Hepatitis B Virus 256
36.1 Clinical Background 256
36.2 Acceptable Specimens 257
36.3 Unacceptable Specimens 257
36.4 Sample Extraction 257
36.5 Primer Sequences 257
36.6 PCR Amplification and Product Detection 258
36.7 Quality Control and Validation Data 258
Reference 259
37 Hepatitis C Virus 260
37.1 Clinical Background 260
37.2 Acceptable Specimens 261
37.3 Unacceptable Specimens 261
37.4 Sample Extraction 262
37.5 Primer Sequences 262
37.6 PCR Amplification and Product Detection 262
37.7 Quality Control and Validation Data 263
Reference 263
38 Hepatitis D Virus 264
38.1 Clinical Background 264
38.2 Acceptable Specimens 265
38.3 Unacceptable Specimens 265
38.4 Sample Extraction 265
38.5 Primer Sequences 265
38.6 PCR Amplification and Product Detection 265
38.7 Quality Control and Validation Data 266
Reference 266
39 Hepatitis E Virus 267
39.1 Clinical Background 267
39.2 Acceptable Specimens 268
39.3 Unacceptable Specimens 268
39.4 Sample Extraction 268
39.5 Primer Sequences 268
39.6 PCR Amplification and Product Detection 268
39.7 Quality Control and Validation Data 269
Reference 269
40 Herpes Simplex Virus Type 1 and 2 270
40.1 HSV Conventional PCR Assay 270
40.1.1 Summary of Methods 270
40.1.2 Acceptable Specimens 270
40.1.3 Sample Extraction 271
40.1.4 Primer and Probe Sequences 271
40.1.5 PCR Amplification and Product Detection 271
40.1.6 Quality Control and Validation Data 272
40.1.7 Assay Limitations 272
40.2 HSV Real-Time PCR Assay 273
40.2.1 Summary of Methods 273
40.2.2 Acceptable Specimens 273
40.2.3 Sample Extraction 273
40.2.4 Primers and Probe Sequences 273
40.2.5 PCR Amplification and Product Detection 274
40.2.6 Quality Control and Validation Data 274
References 274
41 Human Bocavirus 276
41.1 Summary of Methods 276
41.2 Acceptable Specimens 276
41.3 Sample Extraction 276
41.4 Primers and Probe Sequences 276
41.5 PCR Amplification and Product Detection 277
41.6 Quality Control and Validation Data 277
References 277
42 Human Coronaviruses 278
42.1 Summary of Methods 278
42.2 Acceptable Specimens 278
42.3 Sample Extraction 278
42.4 Primer and Probe Sequences 278
42.5 PCR Amplification and Product Detection 279
42.6 Quality Control and Validation Data 280
References 280
43 Human Herpes Viruses -6, -7 and -8 281
43.1 Acceptable Specimens 281
43.2 Sample Extraction 281
43.3 Primer Sequences 281
43.4 Composition of Primer Mixes 283
43.5 PCR Amplification and Product Detection 283
References 283
44 Human Papillomavirus 284
44.1 Summary of Methods 284
44.2 Acceptable Specimens 284
44.3 Unacceptable Specimens 284
44.4 Sample Extraction 284
44.5 Primers and Probe Sequences 285
44.6 PCR Amplification and Product Detection 285
44.7 Quality Control and Validation Data 287
44.8 Assay Limitations 287
References 288
45 Human Polyomaviruses JCV and BKV 289
45.1 Summary 289
45.2 Introduction 289
45.3 JCV and BKV Block PCR Assay 290
45.3.1 Principle of Procedure 290
45.3.2 Sample Extraction 291
45.3.3 Equipment 291
45.3.4 PCR Amplification 291
45.3.5 Product Detection by Agarose Gel Electrophoresis 292
45.3.6 Product Detection Using PCR-ELISA 292
45.3.7 Quality Control 293
45.4 JCV and BKV Real-Time PCR Assay 293
45.4.1 Summary of Methods 293
45.4.2 Acceptable Specimens 294
45.4.3 Sample Extraction 294
45.4.4 Primer and Probe Sequences 294
45.4.5 PCR Amplification and Product Detection 294
45.4.6 Quality Control and Validation Data 295
45.4.7 Assay Limitations 295
References 295
46 Human Polyomaviruses KIV and WUV 296
46.1 Summary of Methods 296
46.2 Acceptable Specimens 296
46.3 Sample Extraction 296
46.4 Primers and Probe Sequences 297
46.5 PCR Amplification and Product Detection 297
46.6 Quality Control and Validation Data 298
References 298
47 Human Rhinoviruses 299
47.1 Background 299
47.2 Acceptable Specimens 299
47.3 Sample Extraction 299
47.4 Primers and Probe Sequences 300
47.5 PCR Amplification and Product Detection 300
47.6 Quality Control and Validation Data 300
47.7 Assay Limitations 300
Reference 301
48 Influenza Virus A H5N1 (Avian Influenza) 302
48.1 Acceptable Specimens 302
48.2 Sample Extraction 302
48.3 Primer and Probe Sequences 303
48.4 PCR Amplification and Product Detection 303
48.5 Quality Control and Validation Data 303
48.6 Assay Limitations 304
References 304
49 Influenza Virus A H1N1 (2009) (Human Swine Influenza) 305
49.1 Summary of Methods 305
49.2 Acceptable Specimens 305
49.3 Sample Extraction 305
49.4 Primers and Probe Sequences 306
49.5 PCR Amplification and Product Detection 306
49.6 Quality Control and Validation Data 306
49.7 Assay Limitations 307
References 307
50 Influenza Type C 308
50.1 Summary of Methods 308
50.2 Acceptable Specimens 308
50.3 Unacceptable Specimens 308
50.4 Sample Extraction 308
50.5 Primers and Probe Sequences 309
50.6 PCR Amplification and Product Detection 309
50.7 Quality Control and Validation Data 309
References 309
51 Measles Virus 310
51.1 Summary of Methods 310
51.2 Acceptable Specimens 310
51.3 Ultracentrifugation 310
51.4 Sample Extraction 311
51.5 Primers and Probe Sequences 311
51.6 PCR Amplification and Product Detection 311
51.7 Quality Control and Validation Data 312
References 312
52 Norovirus 313
52.1 Summary of Methods 313
52.2 Organism 313
52.3 Sample Preparation and RNA Extraction 314
52.4 Primer Sequences 314
52.5 PCR Amplification and Product Detection 315
52.5.1 Nested RT-PCR Assay 315
52.5.2 Single Step RT-PCR Assay 315
References 316
53 Respiratory Syncytial Virus Types A and B 317
53.1 Summary of Methods 317
53.2 Acceptable Specimens 317
53.3 Sample Extraction 317
53.4 Primers and Probe Sequences 318
53.5 PCR Amplification and Product Detection 318
53.6 Quality Control and Validation Data 318
Reference 319
54 Rotavirus 320
54.1 Summary of Methods 320
54.2 Organism 320
54.3 Sample Preparation and RNA Extraction 321
54.3.1 VP7 Genotyping Assay (G Typing) 321
54.3.1.1 VP7 Primer Sequences 321
54.3.1.2 VP7 PCR Amplification and Product Detection 322
54.3.2 VP4 Genotyping (P Typing) 322
54.3.2.1 VP4 Primer Sequences 322
54.4 PCR Amplification and Product Detection 323
54.5 Assay Limitations with Strain Genotyping 324
References 324
55 Varicella Zoster virus 325
55.1 Summary of Methods 325
55.2 Accepted Specimens 326
55.3 Sample Extraction 326
55.4 Primer and Probe Sequences 326
55.5 PCR Amplification and Product Detection 326
55.6 Quality Control and Validation Data 327
References 328
56 Eight Commonly Recognised Respiratory Viruses 329
56.1 Summary of Methods 329
56.2 Acceptable Specimens 329
56.3 Sample Extraction 329
56.4 Primers and Probe Sequences 329
56.5 PCR Amplification and Product Detection 332
56.6 Quality Control and Validation Data 332
56.7 Assay Limitations 332
References 333
57 Detection of CMV, HSV, VZV, EBV and Enterovirus by Multiplex PCR 334
57.1 Principle of Procedure 335
57.2 Specimen Collection 335
57.3 Sample Extraction 335
57.4 Primer and Probe Sequences 336
57.5 PCR Amplification and Product Detection 337
57.5.1 First Round PCR Reaction Mix 337
57.5.2 First Round PCR Amplification 337
57.5.3 Second Round PCR Reaction Mix 337
57.5.4 Second Round PCR Amplification 337
57.6 Agarose Gel Electrophoresis 338
57.7 EIA (DIG) Detection of PCR Products 338
Reference 338
Part V PCR Protocols for Fungal and Parasitic Pathogens 339
58 Aspergillus Species 340
58.1 Summary of Methods 340
58.2 Acceptable Specimens 340
58.3 Unacceptable Specimens 340
58.4 Sample Extraction 341
58.5 Primers and Probe Sequences 341
58.6 PCR Amplification and Product Detection 341
58.7 Quality Control and Validation Data 342
58.8 Assay Limitations 342
References 343
59 Cryptosporidium 344
59.1 Summary of Methods 344
59.2 Organism 344
59.3 Acceptable Specimens 345
59.4 Sample Extraction 345
59.5 Primer and Probe Sequences 345
59.6 Nested PCR 345
59.6.1 PCR Amplification and Product Detection 345
59.6.2 Quality Control and Validation Data 345
59.7 Real Time PCR Using FRET Probes 346
59.7.1 Primer and Probe Sequences 346
59.7.2 PCR Amplification and Product Detection 346
59.7.3 Quality Control and Validation Data 346
References 347
60 Cyclospora 348
60.1 Organism 348
60.2 Summary of Methods 348
60.3 Acceptable Specimens 348
60.4 Sample Extraction 349
60.5 Primer and Probe Sequences 349
60.6 PCR Amplification and Product Detection 349
60.7 Quality Control and Validation Data 349
References 349
61 Dientamoeba fragilis 350
61.1 Summary of Methods 350
61.2 Organism 350
61.3 Acceptable Specimens 350
61.4 Sample Extraction 351
61.5 Conventional PCR 351
61.5.1 Primer Sequences 351
61.5.2 PCR Amplification and Product Detection 351
61.6 Real-Time PCR Using TaqMan Probes 351
61.6.1 Primer and Probe Sequences 351
61.6.2 PCR Amplification and Product Detection 351
61.6.3 Quality Control and Validation Data 352
References 352
62 Entamoeba histolytica 353
62.1 Summary of Methods 353
62.2 Organism 353
62.3 Acceptable Specimens 354
62.4 Sample Extraction 354
62.5 Conventional PCR 354
62.5.1 Primer Sequences 354
62.5.2 PCR Amplification and Product Detection 354
62.6 Conventional PCR Adapted for SYBR Green Real-Time PCR 355
62.6.1 Primer Sequences 355
62.6.2 PCR Amplification and Product Detection 355
62.7 Real Time PCR Using TaqMan Probes 355
62.7.1 Primer Sequences 355
62.7.2 PCR Amplification and Product Detection 355
62.7.3 Quality Control and Validation Data 356
References 356
63 Giardia 358
63.1 Summary of Methods 358
63.2 Organism 358
63.3 Acceptable Specimens 359
63.4 Sample Extraction 359
63.5 Nested PCR 359
63.5.1 Primer Sequences 359
63.5.2 PCR Amplification and Product Detection 359
63.5.3 Quality Control and Validation Data 360
63.6 Real Time PCR 360
63.6.1 Primer Sequences 360
63.6.2 PCR Amplification and Product Detection 360
63.6.3 Quality Control and Validation Data 360
References 360
64 Malaria (P. falciparum, P. vivax, P. malariae, P. ovale) 362
64.1 Summary of Methods 362
64.2 Acceptable Specimens 362
64.3 Sample Extraction 362
64.4 Primers and Probe Sequences 363
64.5 PCR Amplification and Product Detection 363
64.6 Quality Control and Validation Data 363
64.7 Assay Limitations 364
References 364
65 Microsporidia 365
65.1 Summary of Methods 365
65.2 Organism 365
65.3 Acceptable Specimens 365
65.4 Sample Extraction 366
65.5 Conventional PCR 366
65.5.1 Primer Sequences 366
65.5.2 PCR Amplification and Product Detection 366
65.5.3 Quality Control and Validation Data 366
65.6 Real Time Multiplex PCR 367
65.6.1 Primer and Probe Sequences 367
65.6.2 PCR Amplification and Product Detection 367
65.6.3 Quality Control and Validation Data 367
References 367
66 Pneumocystis jirovecii 369
66.1 Background 369
66.2 Acceptable Specimens 369
66.3 Sample Extraction 370
66.4 Primers and Probe Sequences 370
66.5 PCR Amplification and Product Detection 370
66.6 Quality Control and Validation Data 371
66.7 Assay Limitations 371
References 371
67 Trichomonas vaginalis 372
67.1 Summary of Methods 372
67.2 Acceptable Specimens 372
67.3 Sample Extraction 372
67.4 Primer and Probe Sequences 372
67.5 PCR Amplification and Product Detection 373
67.6 Quality Control and Validation Data 373
67.7 Assay Limitations 374
References 374
68 Toxoplasma gondii 375
68.1 Summary of Methods 375
68.2 Acceptable Specimens 375
68.3 Unacceptable Specimens 376
68.4 Sample Extraction 376
68.5 Primer and Probe Sequences 376
68.6 PCR Amplification and Product Detection 376
68.7 Quality Control and Validation Data 377
68.8 Assay Limitations 377
References 377
69 Multiplex PCR for Protozoan Parasites 379
69.1 Summary of Methods 379
69.2 Primer and Probe Sequences: 380
69.3 PCR Amplification and Product Detection 380
69.4 Quality Control and Validation Data 381
References 381
70 Universal Detection and Identification of Fungi by PCR and DNA Sequencing 382
70.1 Summary of Methods 382
70.2 Acceptable Specimens 382
70.3 Unacceptable Specimens 383
70.4 Sample Extraction 383
70.5 Primer Sequences 383
70.6 PCR Amplification and Product Detection 383
70.7 DNA Sequencing 384
70.8 Sequence Assembly and Editing 384
70.9 Quality Control and Validation Data 384
70.10 Assay Limitations 385
References 385
Part VI Susceptibility Screening 386
71 An Introduction to Antimicrobial Susceptibility Screening 387
References 389
72 PCR Assays in Detecting Methicillin Resistance in Staphylococci: Coagulase Negative Staphylococci (CNS), S. aureus, and MRSA with the PVL Gene 390
72.1 Introduction 390
72.2 Single Primer Assay Specific Targeting the mecA Gene for Methicillin Resistance in Both S. aureus and CNS 391
72.2.1 Gel-Based Single Primer Assay for the mecA Gene 391
72.2.1.1 PCR Amplification and Product Detection 391
72.2.2 Real-Time Single Primer Probe Based Assay Targeting the mecA Gene 391
72.2.2.1 PCR Amplification and Product Detection 391
72.2.3 Real-Time Single Primer SYBR Green Based with a Melting Curve Analysis for the mecA Gene 391
72.2.3.1 PCR Amplification and Product Detection 392
72.3 Duplex Primer Assays for MRSA Detection S. aureus Identification for the nuc or femA Gene and Detection of the mecA for Methicillin Resistance 392
72.3.1 Duplex Primer Gel-Based Assay for nuc and mecA Gene Using a Traditional Thermocycler 392
72.3.1.1 PCR Amplification and Product Detection 392
72.3.2 Duplex Real-Time DNA Probe Based Assay for mecA and femA Gene 393
72.3.2.1 PCR Amplification and Product Detection 393
72.3.3 Real-Time Duplex SYBR Green Assay with a Melting Curve Analysis for mecA and nuc Gene 394
72.3.3.1 PCR Amplification and Product Detection 394
72.4 Triplex Real-Time Probe Assay for MRSA and the PVL (Panton-Valentine Leukocidin) Virulence Genes 395
72.4.1 PCR Amplification and Product Detection 395
72.4.2 Quality Control Strains 395
72.4.2.1 Master Mix 395
72.5 Commercial Assays 396
References 396
73 Detection of VRE: vanA and vanB Genes by PCR 398
73.1 Introduction 398
73.2 Real-Time Analysis of vanA and vanB Genes in Enterococcal Isolates by SYBR Green and Melting Curve Analysis 399
73.2.1 Primer Sequences 399
73.2.2 Reaction Mixture 399
73.2.3 Amplification Profile 399
73.2.4 Melt Curve Settings 399
73.2.5 Results 400
73.2.6 Quality Control 400
73.3 The vanA and vanB VRE Gene Light Cycler Assay for Use with Rectal Swabs or Stool Samples Incubated in Enterococcosel Broth 400
73.3.1 Introduction 400
73.3.2 Specimens 401
73.3.3 Materials and Reagents Required 401
73.3.4 Quality Control Strains 401
73.3.5 Extraction Procedures 401
73.3.5.1 VRE Broths 401
73.3.5.2 Control Strains 401
73.3.5.3 Test Organism 402
73.3.5.4 Amplification Procedures 402
73.3.5.5 Interpretation and Reporting van gene PCR Results: Interpretation of Light Cycler Data 402
73.3.6 Reagent Preparation for van gene PCR 403
73.3.6.1 Van Gene PCR Master Mix 403
References 404
74 Metallo Lactamases Gene blaimp, blaspm and blavim Detection by Multiplex Real-Time TaqMan Assay on the Smartcycler 405
74.1 Introduction 405
74.2 Summary of Methods 406
74.3 Acceptable Specimens 406
74.4 Sample Extraction 406
74.5 MBL Multiplex PCR Master Mix 406
74.6 Reactants for 1x Mix 407
74.7 Primers and Probe Sequences 407
74.8 Addition to Reaction Tubes 408
74.9 PCR Amplification and Product Detection 408
74.10 Interpretation and Reporting of PCR Results 408
74.11 Quality Control and Validation Data 408
References 409
75 PCR-Sequencing for Detection of Human Cytomegalovirus Mutations Conferring Antiviral Resistance 410
75.1 Introduction 410
75.2 Specimens 411
75.3 DNA Extraction 411
75.4 PCR Conditions 411
75.5 Analysis of PCR Products 412
References 413
Index 415