Rapid Microarray-Based Genotyping of Chlamydia spp. Strains from Clinical Tissue Samples


Genotype

Known hosts

Subtypes

Type strain (representative strain of subtype)

GenBank acc. no.

A

Psittacine, pigeon, canary, turkey

A-VS1

VS1, MN Zhang

AF269281
 
pheasant, chicken, duck, cattle

A-6BC

6BC

X56980
 
swine, sheep, horse, human

A-8455

84-55

Y16561

B

Pigeon, canary, budgerigar, chicken, pheasant, turkey
 
CP3

AF269265

C

Duck, goose, swan, sheep, human
 
GR9, avian type C

L25436

D

Turkey, human

D-NJ1

NJ1

AF269266
  
D-9 N

9 N

EF375557

E

Pigeon, duck, turkey, ostrich, human
 
CPMN, EAE A22/M

X12647

EB

Duck, human

EB-E30

WS/RT/E30

AY762613
  
EB-859

06-859/1

EU159263
  
EB-KKCP

KKCP-1

AB284062

F

Psittacine
 
VS225

AF269259

M56

Muskrat, snowshoe hare
 
M56

AF269268

WC

Cattle
 
WC

AF269269

1 Va

Crow
 
1 V

EF028916

6 Na

Crow
 
6 N

EF197820

Mat116a

Psittacine, budgerigar
 
Mat116

AB284058

R54a

Antarctic skua
 
R54

AJ243525

YP84a

Psittacine
 
Daruma-1981

AB284065

CPX0308a

Stork
 
CPX0308

AB284064


aAdditional genotypes proposed by Sachse and colleagues [14]



Based on the original serotyping scheme [11], an analogous ompA-based genotyping system has also been in use for strains of the human pathogen C. trachomatis The agent can also be found occasionally in animals [12, 13]. There are 17 generally recognized genotypes, i.e., A, B, Ba, C (associated with trachoma), D, Da, E, F, G, H, I, Ia, J, K (urogenital infections), L1, L2, and L3 (lymphogranuloma venereum). Like in the case of C. psittaci, they are equivalent to the previously defined serotypes.



1.2 Genotyping Using DNA Microarrays


In principle, sequencing of the ompA gene can also be used to identify the genotype of C. psittaci, as well as C. trachomatis. However, there are no generally agreed criteria in terms of sequence similarity, so that the assignment remains somewhat arbitrary. Detailed analysis of ompA sequence data deposited at the GenBank database led Sachse et al. [14] to the conclusion that at least 15 ompA genotypes of C. psittaci are occurring in nature. A summary of this classification in Table 1 shows that the more heterogeneous types A, D, and E/B can be further divided into subtypes. The host range of the proposed new genotypes 1V, 6N, Mat116, R54, YP84, and CPX0308 is not yet fully known, but they seem to occur predominantly in wildlife birds.

The fact that genotype-specific sites of C. psittaci are located in ompA’s variable domains (VD) 2 and 4 allows a molecular definition of individual genotypes at the nucleotide level.

In this situation, the use of DNA microarray technology can provide added value because of its potential to simultaneously exploit minor sequence differences at multiple target sites. In the present chapter, we describe a rapid genotyping assay using the ArrayStrip™ (AS) platform that was shown to work with clinical samples under the conditions of routine diagnosis [14, 15]. The AS platform is open and flexible, so that iterative adaptation of probes and extension beyond the currently covered genotypes will always be possible, for instance, in the case of newly emerging types. Comments on the platform’s major performance parameters and comparison with PCR-RFLP and ompA sequencing are given in Note 1 . While both sensitivity and specificity of the microarray assay are high, its main assets include rapidity, ease of operation, and the possibility of mixed genotype identification, as well as high-throughput and moderate costs. An AS unit consists of eight connected plastic vessels in microtiter format, each of which carrying a microarray chip on the bottom.

The present genotyping procedure is based on target DNA being amplified using duplex PCR with 5′-biotinylated primers to generate a 418-bp product encompassing VD1 and VD2, as well as a 570-bp fragment covering VD3 and VD4. Subsequently, the amplification products are subjected to hybridization on the microarray. Based on the analysis of ompA sequences described above, 35 hybridization probes derived from the discriminatory sites of VD2 and VD4 were selected. They had an average size of 26 nt (22–30), average melting temperature of 60.3 °C (59.7–61.2), and G + C contents of 46.0 mol % (37.0–59.0).

Similarly, an ompA genotyping microarray assay was developed for C. trachomatis [16]. Variable domains 1, 2, and 4 of the ompA locus were amplified using a multiplex PCR with biotinylated primers. A total of 61 oligonucleotide probes representing genotype-specific polymorphisms in the same variable domains were included.



2 Materials



2.1 DNA Extraction


Commercially available DNA extraction kit for PCR template preparation. We use the High Pure PCR Template Preparation Kit (Roche Diagnostics, Mannheim, Germany) for cultured strains and nearly all kinds of tissue samples, e.g., nasal, vaginal, and conjunctival swabs, mucus, bronchoalveolar lavage, organs, feces, and milk.


2.2 Biotinylation PCR and Agarose Gel Electrophoresis




1.

Water. Deionized water must be used throughout.

 

2.

PCR reagents. We use the QIAGEN Multiplex PCR Kit (QIAGEN, Hilden, Germany) according to the instructions of the manufacturer (see Note 2 ).

 

3.

Primers. Sequences, primer concentrations, and other parameters are given in Table 2. The oligonucleotides are stored at −20 °C in 100 μM stock solutions. The concentration of primer working solutions is 10 μM.


Table 2
Primers for ompA genotyping of C. psittaci and C. trachomatis















































Designation

Nucleotide sequence (5′-3′)a

Amount per reaction (μL)b

Amplicon size (bp)

C. psittaci
     

VD1-fw

5′-ACT ACG GAG ATT ATG TTT TCG ATC GTG T-3′

1

418 bp

VD2-rev

5′-Bio-CGT GCA CCY ACG CTC CAA GA-3′

1
 

201CHOMP

5′-GGI GCW GMI TTC CAA TAY GCI CAR TC-3′

2

570 bp

ompA-rev

5′-Bio-TCC TTA GAA TCT GAA TTG AGC-3′

2
 

C. trachomatis
     

Trach-VD1-fw

5′-Bio-ACC AAG CCT TAT GAT CGA C-3′

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Mar 17, 2017 | Posted by in GENERAL | Comments Off on Rapid Microarray-Based Genotyping of Chlamydia spp. Strains from Clinical Tissue Samples

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