Microarray-based detection of bacteria by on-chip PCR.

In this chapter, a protocol called on-chip polymerase chain reaction (PCR) is presented for the deoxyribonucleic acid (DNA) microarray-based detection of bacterial target sequences. On-chip PCR combines, in a single step, the conventional amplification of a target with a simultaneous, nested PCR round intended for target detection. While freely diffusible primers are deployed for amplification, the nested PCR is initiated by oligonucleotide primers bound to a solid phase. Thus, on-chip PCR allows the single-step amplification and characterization of a DNA sample as a result of separation in liquid and solid-phase reactions. In contrast to conventional PCR, the reaction is performed directly on the flat surface of a glass slide that holds an array of covalently attached nested primers. The bacterial target DNA is amplified and probed using primers identifying either species-specific sequence regions of ribosomal DNA or unique bacterial target genes, such as virulence or resistance factors. The microarray is produced using standard spotting equipment with an array layout containing a high number of replicates. Fluorescence scanning of on-chip PCR slides allows the rapid detection of the target of interest. The protocol described herein will show how on-chip PCR can be used to detect and precisely identify DNA of bacterial origin.

Evidence for an association between mannose-binding lectin 2 (MBL2) gene polymorphisms and pre-term birth.

PURPOSE: Human mannose-binding lectin, encoded by the MBL2 gene, is an important component of innate immunity and an important regulator of inflammatory processes. MBL2 gene polymorphisms are associated with an increased risk of neonatal infections and some data suggest a relation between the maternal MBL2 genotype and the risk of premature delivery. In this study, we evaluated whether there is an association between the fetal MBL2 genotype and prematurity. METHODS: A microarray-based on-chip PCR method was used to simultaneously detect five common MBL2 polymorphisms (codon 52, 54, 57; promoter -550, -221) in 204 DNA samples isolated from archival blood cards. MBL2 genotypes of infants born before the 36th week of pregnancy (N = 102) were compared to a control group of infants born at term after the 37th week (N = 102). RESULTS: The frequency of the codon 52 polymorphism was significantly higher in the pre-term group compared to the term group (10.8% versus 4.9%, P = 0.04), while the frequency of the codon 54 polymorphism was equal in both groups (11.3% versus 11.8%). Interestingly, carriers of genotypes (O/O) likely conferring deficient MBL plasma levels were more common in the group of premature birth (9.8% versus 2.9%, P = 0.05), while the promoter -550 C/C genotype was underrepresented in the pre-term birth group (24.5% versus 39.2%, P = 0.03). CONCLUSION: Our data add to the knowledge about genetic predisposition to prematurity and suggest that the fetal MBL2 genotype might be an additional genetic factor contributing to the risk of premature delivery.

Microarray-based detection of mannose-binding lectin 2 (MBL2) polymorphisms in a routine clinical setting.

The assessment of allelic variants in the human mannose-binding lectin 2 (MBL2) gene is of great clinical importance in newborns or immune-suppressed patients at high risk for a variety of infections. Here, we present a study on the genotyping accuracy of a DNA microarray-based on-chip PCR method suited for the detection of five different polymorphisms in the MBL2 gene. We tested 153 genomic DNA samples, prepared from archival blood spots on Guthrie cards, for the presence of allelic variants in the human MBL2 gene by the on-chip PCR method and compared the obtained results of three variants to standard DNA capillary sequencing. The genotyping power of the described assay was readily comparable to DNA sequencing (453/459 correct genotype calls in 153 DNA samples; 98.7% accuracy), mainly due to intrinsic technical benefits of microarrays such as high number of test replicates and automated data analysis. This study demonstrates, for the first time, the accuracy and reliability of a microarray-based on-chip PCR genotyping assay for measuring allelic variants in a routine clinical setting.

Microarray-based identification of bacteria in clinical samples by solid-phase PCR amplification of 23S ribosomal DNA sequences.

The rapid identification of the bacteria in clinical samples is important for patient management and antimicrobial therapy. We describe a DNA microarray-based PCR approach for the quick detection and identification of bacteria from cervical swab specimens from mares. This on-chip PCR method combines the amplification of a variable region of bacterial 23S ribosomal DNA and the simultaneous sequence-specific detection on a solid phase. The solid phase contains bacterial species-specific primers covalently bound to a glass support. During the solid-phase amplification reaction the polymerase elongates perfectly matched primers and incorporates biotin-labeled nucleotides. The reaction products are visualized by streptavidin-cyanine 5 staining, followed by fluorescence scanning. This procedure successfully identified from pure cultures 22 bacteria that are common causes of abortion and sterility in mares. Using the on-chip PCR method, we also tested 21 cervical swab specimens from mares for the presence of pathogenic bacteria and compared the results with those of conventional bacteriological culture methods. Our method correctly identified the bacteria in 12 cervical swab samples, 8 of which contained more than one bacterial species. Due to the higher sensitivity of the on-chip PCR, this method identified bacteria in five cervical swab samples which were not detected by the conventional identification procedure. Our results show that this method will have great potential to be incorporated into the routine microbiology laboratory.