-
Notifications
You must be signed in to change notification settings - Fork 0
Expand file tree
/
Copy pathmolecular-typing.html
More file actions
executable file
·194 lines (180 loc) · 10.2 KB
/
molecular-typing.html
File metadata and controls
executable file
·194 lines (180 loc) · 10.2 KB
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
<html lang="en">
<head>
<meta http-equiv="Content-Type" content="text/html; charset=UTF-8">
<meta charset="utf-8">
<title>PMEN :: Pneumococcal Molecular Epidemiology Network</title>
<meta name="description" content="">
<meta name="viewport" content="width=device-width, initial-scale=1">
<!-- Place favicon.ico and apple-touch-icon.png in the root directory -->
<link rel="stylesheet" href="http://netdna.bootstrapcdn.com/bootstrap/3.1.0/css/bootstrap.min.css">
<link rel="stylesheet" href="css/normalize.css">
<link href='http://fonts.googleapis.com/css?family=Open+Sans:400italic,600italic,400,300,600' rel='stylesheet' type='text/css'>
<!-- fonts, icons for bootstrap -->
<link href="http://netdna.bootstrapcdn.com/font-awesome/4.0.3/css/font-awesome.css" rel="stylesheet">
<link href="css/styles.css" rel="stylesheet">
</head>
<body>
<!-- Navigation bar starts -->
<div class="navbar navbar-fixed-top bs-docs-nav top-menu" role="banner">
<div class="container">
<div class="navbar-header">
<a href="index.html" class="brand">
<img src="img/pmen-logo.png">
</a>
<button class="navbar-toggle" type="button" data-toggle="collapse" data-target=".bs-navbar-collapse">
<span class="sr-only">Toggle navigation</span>
<span class="icon-bar"></span>
<span class="icon-bar"></span>
<span class="icon-bar"></span>
</button>
</div>
<nav class="collapse navbar-collapse bs-navbar-collapse" role="navigation">
<ul class="nav navbar-nav pull-right">
<li >
<a href="index.html">
<!-- <i class="fa fa-home blue"> </i> --> Home
</a>
</li>
<li class="dropdown">
<a class="dropdown-toggle" data-toggle="dropdown" href="#">
About <b class="caret"></b>
</a>
<ul class="dropdown-menu">
<li class="">
<a href="history.html" >History</a>
</li>
<li class="">
<a href="members.html">Members</a>
</li>
</ul>
</li>
<li class="dropdown active">
<a class="dropdown-toggle" data-toggle="dropdown" href="#">Clones <b class="caret"></b></a>
<ul class="dropdown-menu">
<li class=""><a href="criteria.html">Criteria & Nomenclature</a></li>
<li class=""><a href="clone-collection.html">Clone Collection</a></li>
<li class=""><a href="map_view.html" >Clone Map</a></li>
<li class="active"><a href="molecular-typing.html">Molecular Typing</a></li>
</ul>
</li>
<li><a href="contact.html"> Contact</a></li>
</ul>
</nav>
</div>
</div>
<!-- Navigation bar ends -->
<div id="content"> <section>
<div class="container content justify">
<header>
<h1>Molecular Typing</h1>
</header>
<div class="desc">
<p>
Clones to be included into the Network will be subjected to PFGE, MLST and PBP fingerprinting to confirm that they differ from previously accepted clones. If macrolide resistant then tests for erm and mef genes will be performed.
</p>
<header>
<h2>Pulsed-field gel electrophoresis (PFGE):</h2>
</header>
<p>
Genomic DNA is prepared in situ in agarose blocks as described previously (<a href="#1">1</a>, <a href="#2">2</a>) and is digested with SmaI (Life Technologies, Gaithersburg Md). The fragments are resolved by PFGE in 1% agarose (SeaKem GTG agarose; BioWhittaker Molecular Applications, Rockland, Maine) in 0.5Xtris-Borate-EDTA buffer at 14°C at 6V/cm in a CHEF-DR III system (Bio-Rad Laboratories, Hercules, California). The parameters in block 1 are an initial pulse time of 1s increased to 30s for 17 hrs, and in block 2, 5s increased to 9s for 6 hrs.
</p>
<header>
<h2>Multilocus sequence typing:</h2>
</header>
<p>
MLST is carried out as described previously (<a href="#3">3</a>). Briefly, internal fragments of the aroE, gdh, gki, recP, spi, xpt and ddl genes are amplified by PCR from chromosomal DNA using the primer pairs described by Enright and Spratt (<a href="#3">3</a>). The amplified fragments are directly sequenced in each direction using the primers that are used for the initial amplification.
</p>
<p>
The sequences at each of the seven loci are then compared with all of the known alleles at that locus. Sequences that are identical to a known allele are assigned the same allele number whereas those that differ from any known allele, even at a single nucleotide site, are assigned new allele numbers. The assignment of alleles at each locus is carried out using the software at the pneumococcal MLST web site. The alleles at each of the seven loci define the allelic profile of each isolate and their sequence type (ST).
</p>
<p>
Allelic profiles are shown as a series of seven integers which correspond to the alleles at each of the loci, in the order aroE, gdh, gki, recP, spi, xpt and ddl. The relatedness between the isolates is represented as a dendrogram, constructed by the unweighted pair-group method with arithmetic averages (UPGMA), from the matrix of pair-wise differences in the allelic profiles. The allelic profiles of the reference isolates of each clone are deposited within the pneumococcal database at the MLST web site.
</p>
<header>
<h2>PBP fingerprinting:</h2>
</header>
<p>
The pbp1a, pbp2b and pbp2x genes were amplified by PCR using methods and primers previously described (<a href="#4">4</a>). Amplified genes were digested with HaeIII+DdeI (pbp1a) and HaeIII+RsaI (pbp2b and pbp2x) and electrophoresed on 3% gels.
</p>
</div><hr>
<header>
<h1><i class="fa fa-pencil lbrown"></i> Referenced</h1>
</header>
<div class="desc">
<ul>
<li>
<p>
<a name="1"></a>1. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=8253973">Lefèvre, J. C., G.
Faucon, A. M. Sicard, A. M. Gasc</a>. 1993. DNA fingerprinting of <em>Streptococcus Pneumoniae</em> strains by pulsed field gel electrophoresis. J. Clin. Micro. 31:2724-2728.
</p>
</li>
<li>
<p>
<a name="2"></a>2. <a href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=8619583">McDougal, L. K., J. K.
Rasheed, J. W. Biddle and F. C. Tenover</a>. 1995. Identification of multiple clones of extended-spectrum cephalosporin-resistant <i> Streptococcus pneumoniae</i> isolates in the United States. Antimicrob. Agents Chemother. 39:2282-2288.
</p>
</li>
<li>
<p>
<a name="3"></a>3. <a href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=9846740">Enright,
M. C. and B. G. Spratt</a>. 1998. A multilocus sequence typing scheme for <i>Streptococcus
pneumoniae</i>: identification of clones associated with serious invasive disease. Microbiology 144:3049-3060.
</p>
</li>
<li>
<p>
<a name="4"></a>4.
<a href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=10608770">Gherardi,
G., C.G. Whitney, R.R. Facklam and B. Beall.</a> 2000. Major related
sets of antibiotic-resistant pneumococci in the United States as determined
by pulsed-field gel electrophoresis and pbp1a-pbp2b-pbp2x-dhf restriction
profiles. J. Infect. Dis. 181:216-229.
</p>
</li>
</ul>
</div>
</div>
</section>
</div>
<footer>
<!------------------ Social Communications ---------------->
<section id="social-footer">
<div class="container">
<div class="row">
<!-- Twitter widget here -->
<div class="col-lg-12 col-md-12">
<h2><i class="fa fa-sitemap lbrown"></i> Site Map</h2>
<div class="copy">
<p>
<ul>
<li><a href="index.html">Home</a></li>
<li>
<a href="members.html">Members</a> /
<a href="history.html" >History</a> /
<a href="members.html">Members</a>
</li>
<li>
<a href="criteria.html">Criteria & Nomenclature</a> /
<a href="clone-collection.html">Clone Collection</a> /
<a href="map_view.html" >Clone Map</a> /
<a href="molecular-typing.html">Molecular Typing</a>
</li>
<li><a href="contact.html">Contact</a></li>
</ul>
<span class="color">PMEN </span> ©
<a href="#">2014</a>
</p>
</div>
</div>
</div>
</div>
</section>
<div class="clearfix"></div>
</footer>
<script src="js/vendor/modernizr-2.6.2.min.js"></script>
<script src="http://ajax.googleapis.com/ajax/libs/jquery/1.10.2/jquery.min.js"></script>
<script type="text/javascript" src="http://code.jquery.com/jquery.min.js"></script>
<script src="http://netdna.bootstrapcdn.com/bootstrap/3.1.0/js/bootstrap.min.js"></script>
<script src="js/custom.js"></script>
</body>
</html>