Guidelines for Optimisation of a Multiplex Oligonucleotide Ligation-PCR for Characterisation of Microbial Pathogens in a Microsphere Suspension Array

نویسندگان

  • Véronique Wuyts
  • Nancy H C Roosens
  • Sophie Bertrand
  • Kathleen Marchal
  • Sigrid C J De Keersmaecker
چکیده

With multiplex oligonucleotide ligation-PCR (MOL-PCR) different molecular markers can be simultaneously analysed in a single assay and high levels of multiplexing can be achieved in high-throughput format. As such, MOL-PCR is a convenient solution for microbial detection and identification assays where many markers should be analysed, including for routine further characterisation of an identified microbial pathogenic isolate. For an assay aimed at routine use, optimisation in terms of differentiation between positive and negative results and of cost and effort is indispensable. As MOL-PCR includes a multiplex ligation step, followed by a singleplex PCR and analysis with microspheres on a Luminex device, several parameters are accessible for optimisation. Although MOL-PCR performance may be influenced by the markers used in the assay and the targeted bacterial species, evaluation of the method of DNA isolation, the probe concentration, the amount of microspheres, and the concentration of reporter dye is advisable in the development of any MOL-PCR assay. Therefore, we here describe our observations made during the optimisation of a 20-plex MOL-PCR assay for subtyping of Salmonella Typhimurium with the aim to provide a possible workflow as guidance for the development and optimisation of a MOL-PCR assay for the characterisation of other microbial pathogens.

برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

منابع مشابه

Rapid oligonucleotide suspension array-based multiplex detection of bacterial pathogens.

A gene-specific microsphere suspension array coupled with 15-plex polymerase chain reaction (PCR) was developed to screen bacterial samples rapidly for 10 strains of bacteria: Shigella spp. (S. flexneri, S. dysenteriae, and S. sonnei), Staphylococcus aureus, Vibrio cholerae (serology O1 and O139), Legionella pneumophila, and Clostridium botulinum (types A, B, and E). Fifteen sets of highly vali...

متن کامل

A rapid multiplex assay for nucleic acid-based diagnostics.

We have developed a rapid (under 4 hours), multiplex, nucleic acid assay, adapted to a microsphere array detection platform. We call this assay multiplex oligonucleotide ligation-PCR (MOL-PCR). Unlike other ligation-based assays that require multiple steps, our protocol consists of a single tube reaction, followed by hybridization to a Luminex microsphere array for detection. We demonstrate the...

متن کامل

METHODS AND PROTOCOLS Amultiplex oligonucleotide ligation-PCR as a complementary tool for subtyping of Salmonella Typhimurium

Subtyping below the serovar level is essential for surveillance and outbreak detection and investigation of Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) and its monophasic variant 1,4,[5],12:i:(S. 1,4,[5],12:i:-), frequent causes of foodborne infections. In an attempt to overcome the intrinsic shortcomings of currently used subtyping techniques, a multiplex oligonucl...

متن کامل

Analytical validation of the tag-it high-throughput microsphere-based universal array genotyping platform: application to the multiplex detection of a panel of thrombophilia-associated single-nucleotide polymorphisms.

BACKGROUND We have developed a novel, microsphere-based universal array platform referred to as the Tag-It platform. This platform is suitable for high-throughput clinical genotyping applications and was used for multiplex analysis of a panel of thrombophilia-associated single-nucleotide polymorphisms (SNPs). METHODS Genomic DNA from 132 patients was amplified by multiplex PCR using 6 primer ...

متن کامل

Applications of multiplex ligation-dependent probe amplification (MLPA) method in diagnosis of cancer and genetic disorders

Introduction: Lots of human diseases and syndromes result from partial or complete gene deletions and duplications or changes of certain specific chromosomal sequences. Many various methods are used to study the chromosomal aberrations including Comparative Genomic Hybridization (CGH), Fluorescent in Situ Hybridization (FISH), Southern blots, Multiplex Amplifiable Probe Hybridisation (MAP...

متن کامل

ذخیره در منابع من


  با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید

برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

عنوان ژورنال:

دوره 2015  شماره 

صفحات  -

تاریخ انتشار 2015