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

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Ruminant Mycoplasmas PCR 239 Table 2 Temperature-Time Profiles of M. bovis Detection Assays oppD/F System uvr C System First round Second round Primers PpMB920-1/2 PpSM5-1/2 MBOUVRC2-L/R Initial denaturation 96°C for 60 s 94°C for 60 s Denaturation 96°C for 15 s 94°C for 45 s 94°C for 30 s Primer annealing 48°C for 60 s 54°C for 60 s 52°C for 30 s Primer extension 72°C for 150 s 72°C for 120 s 72°C for 60 s Final extension 72°C for 180 s 72°C for 180 s Number of cycles 35 30 35 Amplicon size 1911 bp 409 bp 1626 bp 3.2.3. Detection of M. bovis Here we propose two different detection systems based on oppD/F or uvrC target sequences, respectively (see Notes 4 and 5). Experimental parameters and amplicon sizes are given in Table 2. The sensitivity of both detection systems was established during extensive application over several yr (see Note 5). Primer pairs MBOUVRC2-L/R for the uvrC-based PCR and PpMB920-1/2 for the oppD/F-based PCR can both be used in single-step PCR assays, where detection limits will be around 50 colony forming units (cfu). However, the sensitivity of detection can be increased considerably by running a nested PCR using the oppD/F system. After the first round using primer pair PpMB920- 1/2, the products (5 µL of a 1:100 dilution) are subjected to a second amplification with primers PpSM5-1/2. The detection limit of the nested PCR protocol with bulk tank milk samples was found to be 0.85 cfu equivalents/mL (18). Figure 1 shows the results of the nested oppD/F system being used for examination of DNA extracts from tissue samples of an infected calf. 3.2.4. Detection of M. agalactiae Analysis of uvrC genes revealed that there were sufficient differences between M. agalactiae and M. bovis to design primers MAGAUVRC1-L and MAGAUVRC1-R that are specific for M. agalactiae, thus suggesting its utilization as a target in a species-specific PCR assay (16) (see Note 6).The following temperature–time profile should be used in 35 cycles: denaturation at 94 °C for 30 s, annealing at 50 °C for 30 s, extension at 72 °C for 60 s. The size of the amplicon is 1624 bp.
240 Hotzel et al. Fig. 1 Detection of M. bovis from tissue of different lung lobes and fluid samples of an experimentally infected calf using the nested oppD/F assay. For DNA extraction, the High Pure PCR Template Preparation Kit was used. The eluted DNA fraction was processed as described in Subheading 3.1.3.2., steps 15–19. Lane 1, accessory lobe; lane 2, left diaphragmatic lobe; lane 3, right diaphragmatic lobe; lane 4, right apical cranial lobe; lane 5, left apical cranial lobe; lane 6, endo/pericardium; lane 7, cerebrospinal fluid; lane 8, left carpal joint fluid; lane 9, right carpal joint fluid; lane 10, left tarsal joint fluid; lane 11, right tarsal joint fluid; lane 12, reagent control; lane 13, reference strain PG 45 of M. bovis; lane 14, DNA marker, 100-bp ladder. 3.2.5. Detection of M. mycoides subsp. mycoides SC ( MmmSC) The following nested PCR is particularly suitable for the detection of MmmSC from bronchial lavage fluid samples prepared as described under Subheading 3.1.2. The first amplification reaction is done using 5 µL of DNA extract and primer pair SC3NEST1-L/SC3NEST1-R with 35 cycles of 94°C for 30 s, 52°C for 30 s, 72°C for 30 s. The resulting fragment is 716 bp long. The second round uses 1 µL of the product of the first reaction as a template and primer pair SC3VII/SC3IV-S in 35 cycles with the same temperature–time program. For species identification from culture, a single-step amplification with either of the two primer pairs is sufficient. Important general advice is given in Note 7. 3.2.6. Detection of M. mycoides subsp. mycoides LC ( MmmLC) and M. mycoides subsp. capri A detection system for these caprine mycoplasmas was developed on the basis of their lppA genes, which encode a 62-kDa surface lipoprotein (20).
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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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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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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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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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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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176 Gallien 3.3. Specific Isolation
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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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320 Knutsson and Rådström Fig. 3.
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