Multiplexed-tandem PCR (MT-PCR) by AusDiagnostics is a unique patented technology that allows the detection of multiple targets in one sample without compromising analytical sensitivity and specificity.
Benefits and features of MT-PCR:
- Up to 100 targets can be amplified simultaneously
- High specificity
- Allow high-throughput for maximum efficiency
- Real-time quantitation
- Low amount of sample required
- Low number of multiplexed amplification cycles
How MT-PCR works
A 2-step process, MT-PCR can amplify large number of targets simultaneously whilst preserving the relative quantitation between targets.
STEP 1. Reverse transcription and pre-amplification
In the first step, all amplicons are simultaneously amplified in a multiplexed PCR reaction, but this is only carried out for 15 or 18 cycles. Because very little dNTP is consumed in this number of cycles, each PCR is independent of other reactions, thus preserving the relative quantitative between pathogens.
STEP 2. Real-time PCR
The Step 1 product is diluted and divided into a number of real-time PCR reactions, one for each target. Any non-specific amplicons synthesised in Step 1 are not amplified in Step 2 as Step 2 reactions use primers that are nested inside Step 1 primers.
Enriched sample from Step 1 amplified in separate wells
AusDiagnostics MT-PCR technology mentioned in more than 20 publications. Some references are listed below.
Underdiagnosis of Chlamydia trachomatis and Chlamydia psittaci revealed by Introduction of Respiratory Multiplex PCR assay with Chlamydiaceae family Primers Vinita Rane, Kong Khailin, Jackie Williams, Michelle Francis, Despina Kotsanas, Tony M. Korman, Maryza Graham. Diag Microbiol and Inf Dis, Epub 2017 Nov
Evaluation of a Commercial Multiplex PCR for Rapid Detection of Multi Drug Resistant Gram Negative Infections.
Chavada R, Maley M. Open Microbiol J. 2015 Aug 31;9:125-35. doi: 10.2174/1874285801509010125. eCollection 2015.
Rapid identification of Gram-positive pathogens and their resistance genes from positive blood culture broth using a multiplex tandem RT-PCR assay Hazelton BJ, Thomas LC, Unver T, Iredell JR. J Med Microbiol. 2013 Feb;62(Pt 2):223-31. doi: 10.1099/jmm.0.050385-0. Epub 201
Multiplex-tandem PCR for fungal diagnostics.
Lau A, Stanley K, Sorrell T. Methods Mol Biol. 2013;968:195-201. doi: 10.1007/978-1-62703-257-5_15.
Evaluation of Multiplex Tandem Real-Time PCR for Detection of Cryptosporidium spp., Dientamoeba fragilis, Entamoeba histolytica, and Giardia intestinalis in Clinical Stool Samples. Stark D, Al-Qassab SE, Barratt JL, Stanley K, Roberts T, Marriott D, Harkness J, Ellis JT. J Clin Microbiol. 2011 Jan;49(1):257-62. Epub 2010 Nov 3.
Development and validation of a novel molecular biomarker diagnostic test for the early detection of sepsis.
Sutherland A, Thomas M, Brandon RA, Brandon RB, Lipman J, Tang B, McLean A, Pascoe R, Price G, Nguyen T, Stone G, Venter D.
Crit Care. 2011 Jun 20;15(3):R149. doi: 10.1186/cc10274
Rapid semi-automated quantitative multiplex tandem PCR (MT-PCR) assays for the differential diagnosis of influenza-like illness.
Elektra Szewczuk, Kiran Thapa, Terry Anninos, Kenneth McPhie, Geoff Higgins, Dominic E Dwyer, Keith K Stanley and Jonathan R Iredell
BMC Infectious Diseases 2010, 10:113.
Multiplexed tandem PCR: gene profiling from small amounts of RNA using SYBR Green detection
Keith K. Stanley and Elektra Szewczuk Nucleic Acids Research 2005 33(20)
Last updated 06.06.2019