Multiplex PCR Kit Research Articles

Comparative Evaluation of PCR with Commercial Multiplex M. tuberculosis Detection Kit

Background & Objectives: Undiagnosed and mismanaged tuberculosis (TB) continues to fuel the global epidemic. Rapid, accurate and early diagnosis of tuberculosis is a major health concern especially in developing country like India. It is important to develop rapid, sensitive and specific test (i.e. Polymerase chain reaction; PCR) for early diagnosis of tuberculosis because the conventional methods like Ziehl-Neelsen (ZN) smear, lack the sensitivity & specificity, cultures on Lowenstein-Jensen (LJ) media is time consuming and cumbersome techniques. Current study evaluates the significance of In house PCR with commercially available multiplex PCR kit (IS6110 and MPB64 gene targets) in clinical M. tuberculosis samples. Methods: The performance of In-House PCR for the detection of M. tuberculosis (IS6110 & MPB 64 gene target) was compared with multiplex M.tuberculosis PCR kit (Seeplex R MTB ACE Detection Kit; www. Seegene.com). Fifty samples were processed for ZN smear, culture on LJ media and for PCR.Results: Overall combined PCR positivity for both PCR (In House PCR and multiplex M. tuberculosis PCR kit) was observed 76% (38/50) and 70.7% (29/50) in smear negative (S -ve ), culture negative (C -ve ) extra-pulmonary tuberculosis samples. No significant difference was observed between the positivity rate of both PCR (p = 0.832; χ 2 =0.045). However the smear ZN (6%) and culture positivity (14%) in LJ media was observed very low in extra-pulmonary paucibacillary samples.Interpretation & Conclusion: Current study evaluated the significance of In-House PCR (IS6110 & MPB 64 gene target) with multiplex M. tuberculosis PCR kit in rapid diagnosis of tuberculosis in clinical tuberculosis samples particularly in extra-pulmonary smear negatives.

The increasing application of multiplex nucleic acid detection tests to the diagnosis of syndromic infections.

On 14 January 2013, the US Food and Drug Administration (FDA) announced permission for a multiplex nucleic acid test, the xTAG® Gastrointestinal Pathogen Panel (GPP) (Luminex Corporation, USA), which simultaneously detects 11 common viral, bacterial and parasitic causes of infectious gastroenteritis, to be marketed in the USA. This announcement reflects the current move towards the development and commercialization of detection technologies based on nucleic acid amplification techniques for diagnosis of syndromic infections. We discuss the limitations and advantages of nucleic acid amplification techniques and the recent advances in Conformité Européene - in-vitro diagnostic (CE-IVD)-approved multiplex real-time PCR kits for the simultaneous detection of multiple targets within the clinical diagnostics market.

Identification of new primer binding site mutations at TH01 and D13S317 loci and determination of their corresponding STR alleles by allele-specific PCR

Several commercial multiplex PCR kits for the amplification of short tandem repeat (STR) loci have been extensively applied in forensic genetics. Consequently, large numbers of samples have been genotyped, and the number of discordant genotypes observed has also increased. We observed allele dropout with two novel alleles at the STR loci TH01 and D13S317 during paternity testing using the AmpFℓSTR Identifiler PCR Amplification Kit. The lost alleles reappeared when alternative PCR primer pairs were used. A sequence analysis revealed a G-to-A substitution 82 bases downstream of the last TCAT motif of the repeat region at the TH01 locus (GenBank accession: D00269) and a G-to-T substitution 90 bases upstream of the first TATC motif of the repeat region at the D13S317 locus (GenBank accession: G09017). The frequencies of these two point mutations were subsequently investigated in the Chinese population using sequence-specific primer PCR (SSP-PCR), but neither of these mutations was detected in any of the samples tested. In addition, the DNA samples in which the mutations were identified were amplified to type the point mutations by SSP-PCR to determine the corresponding STR alleles at the two loci. Subsequently, the amplified PCR products with different point mutations and STR repeat numbers were directly sequenced because this strategy overcomes the appearance overlapping peaks generated by different STR alleles and accurately characterizes genotypes. Thus, our findings not only provide useful information for DNA databases and forensic identification but also establish an effective strategy for typing STR alleles with primer binding site mutations.

Epithelial‐Mesenchymal Transition in HPV‐Positive and ‐Negative Oropharyngeal Squamous Cell Carcinoma

Objectives: High-risk human papilloma virus (HPV) infection is associated with carcinogenesis in oropharyngeal squamous carcinoma (OSCC), and patients with HPV (+) tumors have significantly favorable prognosis. However, the reason for this good clinical outcome has not been revealed. Epithelial-mesenchymal transition (EMT) causes aggressiveness of cancer cells. We investigated the expression of the EMT markers and analyzed their correlation with HPV status and prognosis to detect the reason HPV (+) OSCCs respond to treatment. Methods: Eighty patients with OSCC were examined in this study. All high-risk HPV infection were determined with the multiplex PCR kit from each formalin-fixed paraffin-embedded pre-treatment sample. We performed immunohistochemical staining for E-cadherin and vimentin and evaluated the results. Expressions of the markers were graded, and the correlation with TNM stages and prognosis was correlated. Results: Twenty-three of the 80 OSCC tissues were shown to be HPV(+). The 5-year survival rate for patients with HPV (+) tumors (79.1%) was higher than for those with HPV (-) tumors (50.7%). Low e-cadherin expression rate was 56.5% in HPV (+) and 19.2 % in HPV (-).( P = 0.019) E-cadherin expression was not correlated with survival rate in either HPV (+) or (-). Vimentin expression was correlated with N-stage in HPV (-). Conclusions: HPV(+) OSCC originally lost their epithelial cell phenotype compared with HPV(-). The EMT enhances aggressive behavior in HPV (-) OSCC but not in HPV (+).

Comparative Evaluation of Six Commercialized Multiplex PCR Kits for the Diagnosis of Respiratory Infections

The molecular diagnosis of respiratory infection can be performed using different commercial multiplex-based PCR kits whose performances have been previously compared individually to those of conventional techniques. This study compared the practicability and the diagnostic performances of six CE-marked kits available in 2011 on the French market, including 2 detecting viruses and atypical bacteria (from Pathofinder and Seegene companies) and 4 detecting only viruses (from Abbott, Genomica, Qiagen and Seegene companies). The respective sensitivity, specificity, accuracy and agreement of each multiplex technique were calculated by comparison to commercial duplex PCR tests (Argene/bioMérieux) used as gold standard. Eighty-eight respiratory specimens with no pathogen (n = 11), single infections (n = 33) or co-infections (n = 44) were selected to cover 9 viruses or groups of viruses and 3 atypical bacteria. All samples were extracted using the NUCLISENS® easyMAG™ instrument (bioMérieux). The overall sensitivity ranged from 56.25% to 91.67% for viruses and was below 50% with both tests for bacteria. The overall specificity was excellent (>94% for all pathogens). For each tested kit, the overall agreement with the reference test was strong for viruses (kappa test >0.60) and moderate for bacteria. After the extraction step, the hands-on time varied from 50 min to 2h30 and the complete results were available in 2h30 to 9 h. The spectrum of tested agents and the technology used to reveal the PCR products as well as the laboratory organization are determinant for the selection of a kit.

Systematic application of multiplex PCR enhances the detection of bacteria, parasites, and viruses in stool samples

To determine whether systematic testing of faecal samples with a broad range multiplex PCR increases the diagnostic yield in patients with diarrhoea compared with conventional methods and a clinician initiated testing strategy. 1758 faecal samples from 1516 patients with diarrhoea submitted to two diagnostic laboratories were tested for viral, bacterial, and parasitic pathogens by Fast-Track Diagnostics multiplex real-time PCR kits and conventional diagnostic tests. Multiplex PCR detected pathogens in 530 samples (30%): adenovirus (51, 3%), astrovirus (95, 5%), norovirus (172, 10%), rotavirus (3, 0.2%), Campylobacter jejuni/coli (85, 5%), Salmonella spp. (22, 1%), Clostridium difficile (72, 4%), entero-haemorrhagic Escherichia coli (21, 1%), Cryptosporidium spp. (3, 0.2%), Entamoeba histolytica (1, 0.1%), and Giardia lamblia (59, 3%). In contrast, conventional testing detected a pathogen in 324 (18%) samples. Using a systematic approach to the diagnosis of gastroenteritis improved diagnostic yield. This enhanced detection with PCR was achieved by a combination of improved detection of individual pathogens and detection of pathogens not requested or unable to be tested by conventional tests. This approach also allowed earlier identification for most pathogens and created a workflow which is likely to adapt well for many diagnostic laboratories.

Comparison of Anyplex II RV16 with the xTAG Respiratory Viral Panel and Seeplex RV15 for Detection of Respiratory Viruses

A novel multiplex real-time PCR approach (Anyplex II RV16 [RV16]; Seegene, South Korea) was compared with a multiplex endpoint PCR kit (Seeplex RV15 ACE detection kit [RV15]; Seegene) and a liquid bead-based assay (xTAG respiratory viral panel [xTAG]; Abbott, United States). Of nasopharyngeal swabs or aspirates and bronchoalveolar lavage fluid samples submitted for RV15 testing, 199 retrospectively collected positive specimens and 283 prospectively collected specimens were further tested with RV16 and xTAG. A true-positive result was defined as a positive result from all three methods or RV16 and xTAG or RV15 and xTAG. For specimens with discrepant results, monoplex PCR and sequencing of the target viruses were performed. In total, 300 virus-positive specimens yielded 386 viruses. When the bocavirus results were excluded, the overall sensitivities of RV16, RV15, and xTAG were 95.2%, 93.3%, and 87.2%, respectively (95% confidence intervals, 93.0 to 97.4%, 90.8 to 95.8%, and 83.8 to 90.6%, respectively). RV16 was more sensitive than xTAG for coronavirus OC43/HKU1 (100% versus 26.1%; P < 0.0001) and adenovirus (100% versus 79.5%; P < 0.01) but was less sensitive than xTAG for rhinovirus/enterovirus (89.4% versus 97.9%; P < 0.05). RV16 demonstrated higher sensitivity than RV15 for the detection of adenovirus (100% versus 82.1%; P < 0.05). The specificities of all three methods ranged from 98.6% to 100%. Sequencing analysis of 64 rhinovirus-positive samples revealed that RV16 accurately differentiated between rhinovirus and enterovirus. RV16 most frequently missed rhinovirus C. In conclusion, the overall sensitivity of RV16 was better than that of xTAG. However, improvement of the sensitivity for rhinovirus is required.

Efficient multiplex simple sequence repeat genotyping of the oomycete plant pathogen Phytophthora infestans

Genotyping is fundamental to population analysis. To accommodate fast, accurate and cost-effective genotyping, a one-step multiplex PCR method employing twelve simple sequence repeat (SSR) markers was developed for high-throughput screening of Phytophthora infestans populations worldwide. The SSR markers reported for this species were evaluated and the twelve most informative and easily scored were selected. To accomplish a single step genotyping procedure, we optimized primers, fluorescent labels and PCR conditions to genotype using a capillary electrophoresis system with four fluorescent labels (FAM, NED, PET and VIC) and a labeled LIZ standard for sizing of the SSR fragments. The results obtained using commercially available multiplex PCR kits on a set of reference isolates were in agreement with that obtained using primer pairs in simplex reactions. In testing on many thousands of isolates, we have found the markers appropriate for resolving distinct multilocus genotypes (MLGs) of isolates of European and wider populations. Here we demonstrate the utility of the assay on a set of 19 reference isolates plus 77 others sampled from The Netherlands and Great Britain. In most isolates one to two alleles were observed at each locus but the presence of three alleles at a single locus in some isolates was consistent with increased ploidy. Methods are presented that are appropriate for the analysis of datasets comprising isolates of mixed ploidy levels. We also report on the direct P. infestans genotyping from infected field material to collect, store and extract pathogen DNA. A critical step in this multiplex method was the standardization of the protocol between two laboratories in The Netherlands and Great Britain. Reference isolates were exchanged and an allele nomenclature and scoring system agreed. Such co-operation is facilitating the genotyping of international collections of P. infestans isolates in wider networks of laboratories and providing the data required to expand an existing international database of pathogen diversity.

Genetic characterization of a population from Cauca Department with 15 STRs markers

This study established allele frequencies and some parameters of forensic interest with 15 autosomal STRs markers in a sample of 172 unrelated individuals from the Department of Cauca – Colombia using the PowerPlex® 16 BIO System (Promega CO) and Qiagen Multiplex PCR (Qiagen) kits. All markers analyzed showed more than 61% of heterozygosity. Penta E and Penta D were the only systems that are not in Hardy Weinberg equilibrium (p<0.0033) after Bonferroni correction. The probabilities of paternity (W), the power of exclusion (PE) and of discrimination (PD) accumulated for all loci analyzed were 0.9999, 0.9999 and >0.9999, respectively. The parameters of forensic interest have values suitable for routine use in forensic genetics.

Development and use of a single-tube four dye, 21plex direct PCR autosomal STR loci amplification assay for human identification and relationship testing

We describe the development and use of single tube multiplex PCR kit in which 21 commonly used autosomal STR loci, together with the amelogenin locus, are amplified. For relationship testing the amplification occurs directly from a single 1mm filter paper disk containing dried blood or saliva, and the loci are amplified using primers with only four different fluorophores. The alleles are separated and visualized using standard capillary DNA sequencers. An allelic ladder comprising the most prevalent alleles of the 21 loci in the Brazilian population is also used to control for electrophoretic mobility.

Identification and determination of antibiotic susceptibilities of Brucella strains isolated from patients in van, Turkey by conventional and molecular methods.

Purpose: Brucellosis is a worldwide zoonotic disease and still constitutes a major public health problem. In this study, we aimed to identify biovars of Brucella strains isolated from clinical specimens taken from brucellosis patients from the Eastern Anatolia region as well determine the susceptibility of these isolates to tigecycline and azithromycin, drugs that may serve as alternatives to the conventional drugs used in the therapy.Materials and methods: Seventy-five Brucella spp. isolates were included in the study. All strains were identified by both conventional and molecular methods. Brucella Multiplex PCR kit (FC-Biotech, Code: 0301, Turkey) and B. melitensis biovar typing PCR kit (FC-Biotech, Code: 0302, Turkey) were used for molecular typing. Antimicrobial susceptibilities of all strains were determined by E-tests.Results: By conventional biotyping, 73 strains were identified as B. melitensis biovar 3 and two strains as B. abortus biovar 3. Molecular typing results were compatible with conventional methods. The MIC50 and MIC90 values of doxycycline were 0.047 and 0.094; tigecycline 0.094 and 0.125; trimethoprim/sulfamethoxazole 0.064 and 0.19; ciprofloxacin 0.19 for both; streptomycin 0.75 and 1; rifampin 1 and 2 and azithromycin 4 and 8. According to the MIC values, doxycycline was found to be the most effective antibiotic, followed by tigecycline, trimethoprim-sulfamethoxazole and ciprofloxacin.Conclusion: Currently recommended antibiotics for the treatment of brucellosis such as doxycycline, rifampin, streptomycin, trimethoprim-sulfamethoxazole and ciprofloxacin were found to be still effective. While our results showed that tigecycline can be used an alternative agent in the treatment of brucellosis, azithromycin has not been confirmed as an appropriate agent for the treatment.

Development of a multiplex PCR system for the simultaneous detection of white spot syndrome virus and hepatopancreatic parvovirus infection

A multiplex PCR kit for simultaneous detection of white spot syndrome virus (WSSV) and hepatopancreatic parvovirus (HPV) was developed and field testing was conducted. A 604-bp target sequence was selected from the vp28 gene of WSSV. A primer set was developed to amplify a 338-bp DNA fragment at the junction of the NS2 and NS1 protein genes of HPV after alignment of eight sequences from different strains. Another internal positive control primer set produced a 139-bp PCR fragment from the β-actin gene by alignment of this gene from Litopenaeus vannamei, Fenneropenaeus chinensis and Penaeus monodon. The detection limits, tested using purified plasmids, for WSSV and HPV were 21.4 and 19.0 copies respectively. The optimum ratio for HPV, WSSV and β-actin was 3:1:1, with an optimum annealing temperature of 57°C. Field test of the multiplex PCR with 170 L. vannamei individuals from 17 aquaculture farms showed 41.8% coinfection with WSSV and HPV, and 40.0% and 3.5% single infection with WSSV and HPV respectively. No virus-free shrimp farm was found. Ten wild catch F. chinensis individuals showed 60% coinfection, and 40% were infected with HPV.

STR-typing of ancient skeletal remains: which multiplex-PCR kit is the best?

AimTo comparatively test nine commercially available short tandem repeat (STR)-multiplex kits (PowerPlex 16, 16HS, ES, ESI17, ESX17, S5 [all Promega]; AmpFiSTR Identifiler, NGM and SEfiler [all Applied Biosystems]) for their efficiency and applicability to analyze ancient and thus highly degraded DNA samples.MethodsFifteen human skeletal remains from the late medieval age were obtained and analyzed using the nine polymerase chain reaction assays with slightly modified protocols. Data were systematically compared to find the most meaningful and sensitive assay.ResultsThe ESI, ESX, and NGM kits showed the best overall results regarding amplification success, detection rate, identification of heterozygous alleles, sex determination, and reproducibility of the obtained data.ConclusionSince application of these three kits enables the employment of different primer sequences for all the investigated amplicons, a combined application is recommended for best possible and – most importantly – reliable genetic analysis of ancient skeletal material or otherwise highly degraded samples, eg, from forensic cases.

The SNPforID Assay as a Supplementary Method in Kinship and Trace Analysis

Objective: Short tandem repeat (STR) analysis using commercial multiplex PCR kits is the method of choice for kinship testing and trace analysis. However, under certain circumstances (deficiency testing, mutations, minute DNA amounts), STRs alone may not suffice. Methods: We present a 50-plex single nucleotide polymorphism (SNP) assay based on the SNPs chosen by the SNPforID consortium as an additional method for paternity and for trace analysis. The new assay was applied to selected routine paternity and trace cases from our laboratory. Results and Conclusions: Our investigation shows that the new SNP multiplex assay is a valuable method to supplement STR analysis, and is a powerful means to solve complicated genetic analyses.

Developmental Validation of AmpFℓSTR® Identifiler® Plus Kit for Forensic Applications

A validation study was performed on the new STR (Short tandem repeat) multiplex PCR Kit for human identification: AmpFℓSTR® Identifiler® Plus PCR Amplification Kit (IDPlus) released from Applied Biosystems. IDPlus contains the same fifteen loci as AmpFℓSTR® Identifiler® PCR Amplification Kit (ID) which is currently widely utilized in forensic DNA analysis. Consequently, IDPlus has the same discrimination ability as ID. According to the manufacturer, this kit has higher sensitivity, more resistant to PCR inhibitors and less background noise in electrophoresis. Thus, IDPlus was expected to be applied to forensic samples difficult to DNA profiling using ID. An applicability of IDPlus to forensic STR analysis was evaluated. Our study confirmed that IDPlus was more sensitive than ID. IDPlus has about 1.4 times to 1.5 times higher peak than ID when using the same PCR cycle number (28) for both kits. While ID has only one PCR protocol, IDPlus has two PCR protocols differing in cycle number: 28 and 29. To clarify the basic ability we compared heterozygous peak height ratio, stutter, intra-color balance, inter-locus balance among IDPlus to both protocols and ID from 94 individual DNA samples. Lower peak height ratio and the larger standard deviation in heterozygous samples were observed when using IDPlus 29-cycle compared to IDPlus 28-cycle. There was no significant difference between ID and IDPlus 28-cycle and between ID and IDPlus 29-cycle about the peak height ratio. Stutter ratios significantly were different in some loci between ID and IDPlus. Although Applied Biosystems supplies one stutter ratio to filter peaks for each locus to IDPlus in spite of having two PCR protocols, it was confirmed that the same stutter ratio could proper filter out stutter peaks for both cycle numbers. With regard to intra-color balance and inter-locus balance, IDPlus 28-cycle tended to be the highest in balance and ID tended to be the lowest in balance across all the samples and colors. Our validation study indicates IDPlus kit is useful in forensic applications, especially for analysis of trace DNA samples when using 29 cycles. However, forensic biologists must be cautious in interpreting DNA profiles obtained using IDPlus 29 cycles, because peak balance in heterozygous samples tended to be imbalanced when using 29 cycles compared to 28 and ID.

Evaluation of the Seeplex® Meningitis ACE Detection Kit for the Detection of 12 Common Bacterial and Viral Pathogens of Acute Meningitis

BackgroundBacterial meningitis is an infectious disease with high rates of mortality and high frequency of severe sequelae. Early identification of causative bacterial and viral pathogens is important for prompt and proper treatment of meningitis and for prevention of life-threatening clinical outcomes. In the present study, we evaluated the value of the Seeplex Meningitis ACE Detection kit (Seegene Inc., Korea), a newly developed multiplex PCR kit employing dual priming oligonucleotide methods, for diagnosing acute meningitis.MethodsAnalytical sensitivity of the kit was studied using reference strains for each pathogen targeted by the kit, while it's analytical specificity was studied using the human genome DNA and 58 clinically well-identified reference strains. For clinical validation experiment, we used 27 control cerebrospinal fluid (CSF) samples and 78 clinical CSF samples collected from patients at the time of diagnosis of acute meningitis.ResultsThe lower detection limits ranged from 101 copies/µL to 5×101 copies/µL for the 12 viral and bacterial pathogens targeted. No cross-reaction was observed. In the validation study, high detection rate of 56.4% was obtained. None of the control samples tested positive, i.e., false-positive results were absent.ConclusionsThe Seeplex Meningitis ACE Detection kit showed high sensitivity, specificity, and detection rate for the identification of pathogens in clinical CSF samples. This kit may be useful for rapid identification of important acute meningitis-causing pathogens.

A primer binding site mutation at the D2S1338 locus resulting in a loss of amplification

An apparent paternal genetic incompatibility at the D2S1338 locus due to false homozygosity is described. It was disclosed after amplification with the AmpFlSTR ® Identifiler ® PCR Amplification Kit and reproducible with the genRES ®MPX-4 DNA-Typing Kit. After application of an alternative primer set an allele *25 could be amplified, which restored Mendelian inheritance between father and child. Sequencing revealed a C > T transition at the fourth position from the 3′ end of the standard forward primer, which potentially caused the allelic loss with the primer sets in the commercial multiplex PCR kits.

Experiences and surprises with PowerPlex ESI 17 and AmpflSTR NGM SElect in routine casework

The introduction of five “new European standard loci” (D1S1656, D2S441, D10S1248, D12S391 and D22S1045) in forensic DNA analysis called for the development of new Multiplex PCR kits. One of the aims was to combine the widespread SGM+ marker combination with the new markers to be amplified in a single kit. In addition, SE33 was also to be included in the new kits. We present practical experiences that have been made since the introduction of PowerPlex ESI 17 and AmpflSTR NGM SElect in our laboratory.

Use of matrix standards for new fluorophores in capillary sequencers

Most laboratories presently carrying out forensic identification and parentage testing use capillary DNA sequencers to separate and visualize the alleles that are produced by multiplex PCR amplification, whether they be of STR, SNP or DIP loci. Because at least four, but usually five, different fluorophores are used simultaneously, each instrument must undergo its own spectral calibration in which a matrix is created and used to correct the overlapping of fluorescence emission spectra of the mixture of dyes. For this users must acquire matrix standards in addition to the multiplex PCR kits and it is not clear what these standards consist of. In our efforts to develop new multiplex STR and DIP amplification kits using different fluorophores from those commonly used, we were obliged to produce new matrix standards to calibrate our multicapillary sequencers, and we describe how this was done.

Abstract 3070: Simultaneous amplification of multiple genetic/epigenetic tumor markers in a single reaction for high throughput genotyping and aberrant methylation profiling

Abstract Matching appropriate therapeutic regimes with tumor genotypes and epigenotypes is of great medical and economical importance. A prerequisite for this approach is thorough and accurate genotype and epigenotype profiling of tumor specimens, which should be performed in precisely defined homogeneous tumor cell populations with minimum non-tumor cell contaminations. However, this can be very challenging due to the genetic and epigenetic heterogeneities and the scarcity of homogeneous tumor cells within a tumor specimen. Multiplex PCR allows high throughput profiling by maximizing the number of targets/reaction in each sample and can reduce the limitations of such sample shortage. Although several multiplex PCR kits have been commercially available for this purpose for some time, their usefulness is restricted by small amplicon size range, less satisfactory amplification uniformity of different amplicons, and occasional failure in amplifying some amplicons, or false positive amplifications. To solve these problems, we have devised a highly specific, optimization-free multiplex PCR formulation (Applied Biosystems Platinum® Multiplex PCR Master Mix) with novel reaction chemistry. We show here that this new formulation is excellent for simultaneous amplification and high throughput profiling of SNPs, deletions, insertions and their wild types of many targets in a single reaction in terms of robustness, uniformity, amplicon size range, compatibility with amplicon GC contents, and the reduction of non-specific primer dimers and false positive/negative products in multiplex PCR. In combination with a primer system of varied lengths and compositions of 5’ stem-loop tag sequences, we demonstrate that even mixtures of multiple adjacent or overlapping alleles in the same locus of EGFR/KRAS/BRAF mutations can be specifically amplified in the same reaction and distinguished by amplicon size differentiation in a single lane of an agarose gel. Moreover, using primer sets specific for methylated and converted DNA, and those specific for unmethylated and converted DNA, we demonstrate that EGFR/KRAS/BRAF mutations and their associated promoter methylation status can be amplified from bisulfite-treated genomic DNA of colon cancer cell lines in a single reaction. In conclusion, the Applied Biosystems Platinum® Multiplex PCR Master Mix and the stem-loop primer system are a “best in class” solution to simultaneous amplification of various types of genetic and epigenetic tumor markers of multiple targets for high throughput genotyping and aberrant methylation profiling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3070. doi:10.1158/1538-7445.AM2011-3070