Scorpion Probe Research Articles

Assessment of Digital PCR as a Primary Reference Measurement Procedure to Support Advances in Precision Medicine.

Genetic testing of tumor tissue and circulating cell-free DNA for somatic variants guides patient treatment of many cancers. Such measurements will be fundamental in the future support of precision medicine. However, there are currently no primary reference measurement procedures available for nucleic acid quantification that would support translation of tests for circulating tumor DNA into routine use. We assessed the accuracy of digital PCR (dPCR) for copy number quantification of a frequently occurring single-nucleotide variant in colorectal cancer (KRAS c.35G>A, p.Gly12Asp, from hereon termed G12D) by evaluating potential sources of uncertainty that influence dPCR measurement. Concentration values for samples of KRAS G12D and wild-type plasmid templates varied by <1.2-fold when measured using 5 different assays with varying detection chemistry (hydrolysis, scorpion probes, and intercalating dyes) and <1.3-fold with 4 commercial dPCR platforms. Measurement trueness of a selected dPCR assay and platform was validated by comparison with an orthogonal method (inductively coupled plasma mass spectrometry). The candidate dPCR reference measurement procedure showed linear quantification over a wide range of copies per reaction and high repeatability and interlaboratory reproducibility (CV, 2%-8% and 5%-10%, respectively). This work validates dPCR as an SI-traceable reference measurement procedure based on enumeration and demonstrates how it can be applied for assignment of copy number concentration and fractional abundance values to DNA reference materials in an aqueous solution. High-accuracy measurements using dPCR will support the implementation and traceable standardization of molecular diagnostic procedures needed for advancements in precision medicine.

Dual surrogate markers for rapid prediction of epidermal growth factor receptor mutation status in advanced adenocarcinoma of the lung: A novel approach in resource-limited setting.

Tyrosine kinase inhibitors have revolutionized the treatment of metastatic lung cancer in patients with epidermal growth factor receptor (EGFR) mutations. Amplified refractory mutation system (ARMS)-reverse transcription-polymerase chain reaction (RT-PCR), the current standard for detecting EGFR mutation status is time-consuming and highly expensive. Consequently any surrogate test which are cheaper, faster and as accurate as the PCR method will help in early diagnosis and management of patients with lung cancer, especially in resource-limited settings. Eighty-five patients, all of South Indian origin, with adenocarcinoma of lung, registered between October 2009 and January 2013, were evaluated for EGFR mutation status by using scorpion probe based ARMS RT-PCR method. Immunohistochemical (IHC) was performed using the phosphorylated AKT (P-AKT) and thyroid transcription factor-1 (TTF-1) on above patient's sample, and the results were compared with EGFR mutation tests. EGFR mutation was positive in 34 of 85 patients (40%). P-AKT and TTF-1 were positive in 50 (58.8%) and 68 (80%) patients respectively. Both P-AKT and TTF-1 had statistically significant correlation with EGFR mutation status. Positive and negative predictive value of P-AKT in diagnosing EGFR mutation was 58% and 85.5% and that for TTF-1 was 48.5% and 94.1%, respectively. The problem of low positive predictive value can partly be overcome by testing P-AKT and TTF-1 simultaneously. P-AKT and TTF-1 using IHC had statistically significant correlation with EGFR mutation with high negative predictive value. In the case of urgency of starting treatment, EGFR mutation testing may be avoided in those patients who are negative for these IHC markers and can be started on chemotherapy.

A High-Throughput Screening Method to Reengineer DNA Polymerases for Random Mutagenesis

A screening system for directed evolution of DNA polymerases employing a fluorescent Scorpion probe as a reporter has been developed. The screening system has been validated in a directed evolution experiment of a distributive polymerase from the Y-polymerase family (Dpo4 from Sulfolobus solfataricus) which was improved in elongation efficiency of consecutive mismatches. The engineering campaign yielded improved Dpo4 polymerase variants one of which was successfully benchmarked in a sequence saturation mutagenesis experiment especially with regard to the desirable consecutive transversion mutations ([2.5-fold increase in frequency relative to a reference library prepared with Dpo4 WT). The Scorpion probe screening system enables to reengineer polymerases with low processivity and fidelity, and no secondary activities (i.e. exonuclease activity or strand displacement activity) to match demands in diversity generation for directed protein evolution.

Association of PIK3CA kinase domain mutations with poor prognosis in operable breast cancer.

e21060 Background: The PI3K pathway is frequently altered in breast cancer (BC) and may contribute to therapeutic resistance and clinical outcome. However, the findings concerning the impact of PIK3CA mutations on BC are controversial. The aim of this study was to characterize the PIK3CA mutations in a cohort of 101 operable BC patients receiving systemic adjuvant therapy and to determine associations with pathological features and patient outcome. Methods: Mutations in the helical (E542K, E545K) and the kinase (H1047R, H1047L) domains of the PIK3CA gene were investigated by direct sequencing and Amplification Refractory Mutation System (ARMS) allele-specific PCR and Scorpion probes in FFPE tumor samples. The prognostic significance of PIK3CA mutations for disease-free survival (DFS), and overall survival (OS) was evaluated in univariate and multivariate analysis adjusted for the type of surgery (breast-conserving vs mastectomy), adjuvant treatment administered, histological grading, tumor size, axillary node infiltration, menopausal status, estrogen (ER) as well as progesterone (PR) receptor and HER-2 status. Results: PIK3CA mutations were detected in 21(21%) of the primary tumors (E542K in one, E545K in 4, E545D in one, H1047R in 11 and H1047L in 4) and were significantly associated with PR+ BC (p=0.036). After a median follow-up of 60.5 (range 4.6-199) months 27 (27%) patients had relapsed and 18 (18%) had died. PIK3CA kinase domain mutations were associated with significantly shorter DFS (p=0.001) and OS (p=0.012). Multivariate analysis showed that PIK3CA kinase domain mutations, nodal involvement and tumor size were independent prognostic factors for OS. The relative risk of death in patients with PIK3CA kinase domain mutations was 7.68 (95% CI = 2.22-26.57, p=0.001). Conclusions: Our results suggest that PIK3CA kinase domain mutations are an independent risk factor for poor prognosis in operable breast cancer patients.

Assessment of the genetic stability of GMOs with a detailed examination of MON810 using Scorpion probes

For the cultivation and maintenance of seeds, genetic mutations have to be avoided as much as possible. According to the Directive 2001/18/EC, genetic stability has to be proven for the approval of GMOs [1]. The investigation of genetic stability can be performed with various methods, but Southern blots are most commonly used. In this study, a different method, a real-time PCR involving Scorpion primer, was developed for the analysis of DNA rearrangements in GMOs. The advantage of this method is that already small changes in sequence can be detected. In addition, large numbers of samples can be tested at once in a single PCR run. Possible alterations in the region of the Scorpion primer can be detected by differences in the real-time PCR plots. The sequence that was analysed contained 130 nucleotides. The sensitivity of Scorpion primers to measure genetic stability was verified in experiments with plasmids that had only minor sequence alterations in the target region. Such small differences led to a total reduction of the fluorescence signal, even in heterozygous samples where the unaltered template was present as well. Scorpion primers were then used to investigate the transgenic sequences of 567 individual seeds of MON810 for maize at the 5′ and the 3′ region of the insert. In combination with SYBR green, the screening procedure allowed a selection of samples for further analysis. If a sample showed low fluorescence with the Scorpion primer but a normal amplification with SYBR green, then the integrity of the insert was checked by sequencing. The results, however, gave no indication of a mutation or rearrangement of MON810 seeds. In additional experiments, the reliability of the method was studied by determining several parameters that are required for a validation, e.g., specificity, cross-reactivity, dynamic range, correlation of the template concentration and the corresponding Ct values, PCR efficiency, relative repeatability standard deviation (RSDr) relative reproducibility standard deviation (RSDR).

Real‐time PCR (qPCR) primer design using free online software

Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most commonly used fluorescent chemistries are SYBR® Green dyes and TaqMan®, Molecular Beacon or Scorpion probes. SYBR® Green is very simple to use and cost efficient. As SYBR® Green dye binds to any double-stranded DNA product, its success depends greatly on proper primer design. Many types of online primer design software are available, which can be used free of charge to design desirable SYBR® Green-based qPCR primers. This laboratory exercise is intended for those who have a fundamental background in PCR. It addresses the basic fluorescent chemistries of real-time PCR, the basic rules and pitfalls of primer design, and provides a step-by-step protocol for designing SYBR® Green-based primers with free, online software.

Screening for EGFR mutations in lung cancer, a report from India

Epidermal growth factor receptor (EGFR) is one of the targeted molecular markers in many cancers including the lung malignancy. Genetic modifications such as deletions, insertions and Single Nucleotide Polymorphisms in the tyrosine kinase (TK) domain of EGFR is a common feature observed in most lung cancers. Gefitinib and erlotinib are commonly available therapeutic drugs which act as specific inhibitors for the tyrosine kinase domain of EGFR and associated with EGFR mutations in exons 18–21. However the prevalence of mutation varies among ethnicity, grade, age and gender. This is the first report on the prevalence of EGFR mutation in non-small cell lung cancer patients using DNA obtained from samples such as biopsy/cytology/pleural fluid and Fine Needle Aspiration (FNA), across India. We have screened for 29 somatic mutations which span exons 18, 19, 20 and 21 of EGFR gene using Scorpion probe based ARMS-PCR technique. DNA from 220 NSCLC tissue samples were analyzed for EGFR mutations and mutations were detected in 51.8% of the study population. Among the mutant positive cases, the deletions in exon 19 (52%) and a missense mutation L858R in exon 21 (26%) were most predominant. There was a significant increase in overall mutations (p=0.01) as a function of age, mutation in exons 19 and 21 together (p=0.003), mutations in exons 18, 19 and 21 (p=0.04) and mutations in exons 18 and 19 (p=0.03) in females. Mutations did not seem to significantly correlate metastases or disease progression. Mutations in exons [19] and 21 together were significant in non-smokers compared to smokers (p=0.01) using Mann–Whitney tests. The study suggests high prevalence of EGFR positivity in NSCLC in Indian sub-population and provides opportunities for targeted therapies for this group.

Surpassing Specificity Limits of Nucleic Acid Probes via Cooperativity

The failure to correctly identify single nucleotide polymorphisms (SNPs) significantly contributes to the misdiagnosis of infectious disease. Contrary to the strategy of creating shorter probes to improve SNP differentiation, we created larger probes that appeared to increase selectivity. Specifically, probes with enhanced melting temperature differentials (>13x improvement) to SNPs were generated by linking two probes that consist of both a capture sequence and a detection sequence; these probes act cooperatively to improve selectivity over a wider range of reaction conditions. These cooperative probe constructs (Tentacle probes) were then compared by modeling thermodynamic and hybridization characteristics to both Molecular Beacons (stem loop DNA probes) and Taqman probes (a linear oligonucleotide). The biophysical models reveal that cooperative probes compared with either Molecular beacons or Taqman probes have enhanced specificity. This was a result of increased melting temperature differentials and the concentration-independent hybridization revealed between wild-type and variant sequences. We believe these findings of order of magnitude enhanced melting temperature differentials with probes possessing concentration independence and more favorable binding kinetics have the potential to significantly improve molecular diagnostic assay functionality.

Detection of PIK3CA mutations in circulating free DNA in patients with breast cancer

Somatic mutations in PIK3CA (encoding a class I phosphoinositide 3 kinase (PI3K) subunit) modulate PI3K signalling to influence tumour behaviour and occur in up to 40% of breast cancers. Inhibitors of PI3K signalling are entering clinical trials, but the impact of PIKC3A mutation on tumour response has yet to be clarified. This study investigated the potential utility of circulating free DNA (cfDNA) as a source for PIK3CA mutation detection in patients with breast cancer. cfDNA extracted (QIAamp Virus spin kit) from blood and matched archival tumour from 46 patients with metastatic breast cancer and 30 patients with localised, operable breast cancer was assessed for hotspot PIK3CA mutations using Amplification Refractory Mutation System (ARMS()) allele-specific PCR and Scorpion probes. PIK3CA mutations were detected in 13/46 (28%) plasma-derived and 10/46 (21%) serum-derived cfDNA samples from metastatic breast cancer patients. In 41 cases with matched tumour and plasma-derived cfDNA data, concordance (same mutation status in plasma and tumour) was 95%. Where a PIK3CA mutation was present in tumour, the 'pick up' in plasma-derived cfDNA was 80%. PIK3CA mutations were present in tumours from 14/30 (47%) localised breast cancers, but no PIK3CA mutations were detected in matched cfDNA. These data demonstrate feasibility and potential utility of cfDNA for PIK3CA mutation detection in patients with metastatic breast cancer. Studies are underway to qualify PIK3CA mutation in cfDNA as a predictive biomarker allowing patient stratification in clinical trials of mechanism-based therapeutics that target PI3K signalling pathways.

A Scorpion Probe–Based Real-Time PCR Assay for Detection of E. coli O157:H7 in Dairy Products

Methods for rapidly detecting Escherichia coli O157:H7, a high risk foodborne pathogen of considerable public health concern, are extremely important for monitoring of food safety. Conventional detection such as microbiological, biochemical, and immunological methods, including polymerase chain reactions (PCR), are time consuming and only qualitative. In this study, a rapid real-time PCR (RTi-PCR) method based on Scorpion probe technology targeting the eae gene of E. coli O157:H7 was developed and applied using spiked and naturally contaminated products. The assay was highly specific and did not show any cross-reactivity with any of the 15 nontargeted bacterial cultures. The assay was sensitive enough to detect 2 log colony-forming units (CFU)/mL of the target pathogen in pure broth culture and 3 log CFU/mL in spiked skim milk. When the assay was applied with 60 market dairy food samples, one sample each of raw milk, paneer, and ice cream, was found to be positive for E. coli O157:H7 with respective RTi-PCR counts of 6.7, 6.2, and 5.9 log CFU. The present study demonstrates the application of Scorpion probe technology for the detection of foodborne pathogens and reports the presence of E. coli O157:H7 in paneer, a very popular indigenous dairy product (soft cheese) in India, for the first time.

Comparison of genotyping with direct sequencing as a method to detect EGFR mutations in NSCLC

22013 Background: EGFR mutations are detected in 10–15% of patients with non-small cell lung cancer (NSCLC). The most common clinically used method, and used at our institution, of EGFR mutation detection is based on direct DNA sequencing of exons 18–21. Most of the clinically significant EGFR mutations have been characterized and hence an alternative mutation detection method could be appropriate; we explored a genotyping method using scorpion probes (DXs; Manchester, UK). Methods: We compared genotyping (EGFR 29 mutation kit; Dxs) in a blinded fashion using 147 NSCLC specimens that had been previously sequenced in a CLIA (Clinical Laboratory Improvement Amendments) certified sequencing laboratory at Harvard. DNA specimens were from adenocarcinomas and typically had ≥ 50% tumor content. Results: In the 147 specimens, direct sequencing identified 42 (29%) EGFR mutations (Exons 18/19/20/21: 1/22/4/15). Of these, genotyping using scorpion probes (DxS) identified 39 (93%; sensitivity 93%). This method did not detect 3 mutations that were detected by sequencing including 2 exon 19 deletions and 1 exon 20 insertion; 1 of the exon 19 deletions is not assayed for in the DxS 29 mutation EGFR genotyping kit. Genotyping detected 5 additional EGFR mutations (Exon 19 deletion:1; EGFR T790M: 2 and L858R: 2) not detected by direct sequencing (specificity 96%). Four of the 5 (80%) were confirmed using a second methodology (WAVE/Surveyor). One of the 5 patients (L858R) has been treated with erlotinib and her tumor has regressed with treatment. No treatment information is available on the other 4 pts. Conclusions: Genotyping (sensitivity 93%; specificity 96%) provides an alternative method to direct sequencing for EGFR mutation detection. The biological and clinical significance of EGFR mutations detected by genotyping but not detected by direct sequencing is not fully defined. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration AstraZeneca, Boehringer Ingelheim, Roche Pfizer

Target-assembled exciplexes based on Scorpion oligonucleotides

Scorpion probes, specific DNA probe sequences maintained in a hairpin-loop, can be modified to carry the components of an exciplex for use as a novel fluorescence-based method for specific detection of DNA. The exciplex partners (5'-pyrenyl and 3'-naphthalenyl) were attached to oligonucleotides via phosphoramidate links to terminal phosphate groups. Hybridization of the probe to a complementary target in a buffer containing trifluoroethanol produced an obvious fluorescence change from blue (pyrene locally excited state emission) to green (exciplex emission).

A novel fluorescent quantitative polymerase chain reaction method of Mycobacterium tuberculosis DNA using duplex scorpion probe

Objective To establish a new fluorescent probe-based polymerase chain reaction (PCR) assay (the duplex scorpion probe) to the detect Mycobacterium tuberculosis(TB) DNA. MethodsThe recombinant vector pMD18-T-senX3-regX3 IR was used as the standard template. The duplex scorpion probe was designed according to the cloned gene sequence,and then the PCR reaction system was optimized and evaluated.Results (1) The plasmid containing the sequence of interest was constructed successfully. (2) The assay for Mycobacterium tuberculosis was developed with large detecting range, high sensitivity, specificity, stability and accuracy. (3) After applied in clinical experiments, this novel fluorescent quantitative method has more advantages than the Taqman detecting kit. Conclusions A new real-time PCR detective method for mycobacterium tuberculosis has been successfully developed, which would lay a substantial foundation for the development of a new kit’s competition on the market. Our research has showed that the duplex scorpion probe assays are more accurate, sensitive, specific, and low-cost than the Taqman assay, which suggested that the real-time PCR for TB based on duplex scorpion probe was a novel and excellent method for the gene diagnosis of tuberculosis potentially.

Real-time PCR detection and speciation of Cryptosporidium infection using Scorpion probes

At least eight species of Cryptosporidium can cause human infection and disease. A real-time PCR (qPCR) assay based on the 18S rRNA gene and utilizing a Scorpion probe was developed to detect all human-pathogenic Cryptosporidium without the usual need for nested amplification. Sensitivity of detection in stool samples was highest using a glass bead-based DNA extraction method (under 10(3) oocysts per stool sample). The assay was validated against 123 human stool specimens from Bangladesh and Tanzania, exhibited a sensitivity and specificity of >91% versus microscopy, and detected an additional eight microscopy-negative infections. Cryptosporidium parvum-specific and Cryptosporidium meleagridis-specific Scorpion qPCR assays that provided 100% accurate speciation compared with VspI RFLP analysis and sequencing were developed subsequently. These Scorpion probe qPCR assays are simpler to perform than existing nested PCR and RFLP methods for diagnosis and epidemiological investigation of cryptosporidiosis.

Evaluation of probe chemistries and platforms to improve the detection limit of real-time PCR

A validated PCR-based Salmonella method targeting a 94-bp sequence of the ttr gene was used as a model to compare six different combinations of reporter and quencher dyes of a TaqMan probe, on three different instruments, to improve the detection limit in a real-time PCR assay with the aim of a same-day analysis. The use of locked nucleic acids (LNA) and Scorpion probes were also tested. The combination FAM–BHQ1 or Cy5–BHQ3, both dark quenchers, gave the best results (Cycle threshold (Ct) of 25.42 ± 0.65 and 24.47 ± 0.18 at 10 3 DNA copies). When comparing different probe technologies, the LNA probe (FAM–BHQ1) was the most sensitive with the strongest fluorescence signal (dR last 48 066), resulting in 0.6 to 1.1 lower Ct values than a DNA TaqMan probe, and 1.9 to 4.0 lower Ct than the Scorpion system (FAM–BHQ1). The RotorGene real-time PCR instrument gave 0.4–1.0 lower Ct values (more sensitive) than the Mx3005p, and 1.5–3.0 lower than the ABI 7700. Using the LNA in a RotorGene instrument, we detected the following Salmonella DNA copies in 1-ml pre-enriched samples: fishmeal (100 copies), chicken rinse (100 copies) and pig feces (10 copies). The detection probability of the final assay on inoculated fecal samples was 100% at 2 × 10 4 copies per ml. In conclusion, the LNA probe with annealing temperature of 65 °C could be useful for more sensitive detection limits.

Use of Bacteriophage MS2 as an Internal Control in Viral Reverse Transcription-PCR Assays

Diagnostic systems based on reverse transcription (RT)-PCR are widely used for the detection of viral genomes in different human specimens. The application of internal controls (IC) to monitor each step of nucleic acid amplification is necessary to prevent false-negative results due to inhibition or human error. In this study, we designed various real-time RT-PCRs utilizing the coliphage MS2 replicase gene, which differ in detection format, amplicon size, and efficiency of amplification. These noncompetitive IC assays, using TaqMan, hybridization probe, or duplex scorpion probe techniques, were tested on the LightCycler and Rotorgene systems. In our approach, clinical specimens were spiked with the control virus to monitor the efficiency of extraction, reverse transcription, and amplification steps. The MS2 RT-PCR assays were applied for internal control when using a second target hepatitis C virus RNA in duplex PCR in blood donor screening. The 95% detection limit was calculated by probit analysis to 44.9 copies per PCR (range, 38.4 to 73.4). As demonstrated routinely, application of MS2 IC assays exhibits low variability and can be applied in various RT-PCR assays. MS2 phage lysates were obtained under standard laboratory conditions. The quantification of phage and template RNA was performed by plating assays to determine PFU or via real-time RT-PCR. High stability of the MS2 phage preparations stored at -20 degrees C, 4 degrees C, and room temperature was demonstrated.

Detection of Meloidogyne incognita and Pochonia chlamydosporia by fluorogenic molecular probes*

Fluorescent molecular probes were applied for detection of the plant parasitic nematode Meloidogyne incognita and the nematode‐egg parasitic fungus Pochonia chlamydosporia var. chlamydosporia. A region in the M. incognita rDNA including ITS2 was selected for amplification and recognition with a real‐time PCR assay, based on a combination of three specific motifs, each recognized by a specific fluorescent probe. Similarly, a Scorpion probe was designed for the RT‐PCR quantification of P. c. chlamydosporia. For this purpose, the ITS‐2 rDNA gene of the fungus was sequenced from a number of Italian isolates. A conserved region unique for P. c. chlamydosporia found in the ITS‐2 rDNA gene was used. The probes allowed recognition of single juveniles of M. incognita and of the mycelium‐ or soil‐extracted fungal DNA. The potentialities of the detection procedures are discussed.

Isolate-Specific Detection of Grapevine fanleaf virus from Xiphinema index Through DNA-Based Molecular Probes

ABSTRACT Tests with a real-time reverse transcription-polymerase chain reaction (RT-PCR) were performed on specimens of Xiphinema index collected from the rhizosphere of Grapevine fanleaf virus (GFLV)-infected grapevines at Palagiano, Italy. A 1,157-bp fragment of the GFLV RNA-2 coat protein (CP) gene was amplified and sequenced. A fluorescent Scorpion probe was designed to detect a highly conserved CP region. A second region with isolate-specific multiple nucleotide polymorphisms was used to detect GFLV isolates using molecular beacons (MB). The Scorpion probe allowed quantitative estimation of GFLV RNA-2 in single nematodes, using a dilution series of a 692-nucleotide transcript of the CP gene. The assay allowed detection of GFLV RNA-2 in individual X. index, with a minimum template threshold of 800 fg or 2.8 x 10(6) RNA-2 molecules per nematode. The CP fragment used for GFLV detection with the Scorpion probe appeared highly conserved among isolates. The probes were tested against other GFLV isolates, which were recognized by the species-specific Scorpion probe and by the corresponding MB specific to the particular isolate. Both tests appeared useful as diagnostic tools or for studies on GFLV in acquisition, retention, and transmission experiments.

Basic principles of real-time quantitative PCR

Real-time quantitative PCR allows the sensitive, specific and reproducible quantitation of nucleic acids. Since its introduction, real-time quantitative PCR has revolutionized the field of molecular diagnostics and the technique is being used in a rapidly expanding number of applications. This exciting technology has enabled the shift of molecular diagnostics toward a high-throughput, automated technology with lower turnaround times. This article reviews the basic principles of real-time PCR and describes the various chemistries available: the double-stranded DNA-intercalating agent SYBR® Green 1, hydrolysis probes, dual hybridization probes, molecular beacons and scorpion probes. Quantitation methods are discussed in addition to the competing instruments available on the market. Examples of applications of this important and versatile technique are provided throughout the review.

Multiplex real-time PCR assay using Scorpion probes and DNA capture for genotype-specific detection of Giardia lamblia on fecal samples.

Two major genotypic assemblages of Giardia lamblia infect humans; the epidemiologic significance of this phenomenon is poorly understood. We developed a single-vessel multiplex real-time PCR (qPCR) assay that genotypes Giardia infections into assemblages A and/or B directly from fecal samples. The assay utilized Scorpion probes that combined genotype-specific primers and probes for the 18S rRNA gene into the same molecule. The protocol was capable of detecting as few as 20 trophozoites per PCR on fecal DNA isolated using a commercial method or 1.25 trophozoites per PCR on fecal DNA isolated using a G. lamblia-specific oligonucleotide capture technique. The assay was specific for fecal specimens, with no amplification of the discordant genotype with the opposite Scorpion probe. When 97 clinical specimens from Bangladesh were used, the multiplex PCR assay detected 95% (21 of 22) of Giardia microscopy-positive specimens and 18% (13 of 74) of microscopy-negative specimens. Microscopy-negative and qPCR-positive specimens had higher average cycle threshold values than microscopy-positive and qPCR-positive specimens, suggesting that they represented true low-burden infections. Most (32 of 35) infections were assemblage B infections. This single-reaction multiplex qPCR assay distinguishes assemblage A Giardia infections from assemblage B infections directly on fecal samples and may aid epidemiologic investigation.