Duplex ddPCR Research Articles

Development of the duplex droplet digital PCR for quantitative monitoring of mixed infections of Meloidogyne incognita and M. enterolobii.

Root-knot nematodes (RKN, Meloidogyne spp.) are economically significant pests that cause immense damage to a wide range of crops. Among them, M. incognita and M. enterolobii are of particular concern, as their high virulence and broad host range. RKN are challenging for detection due to their subterranean lifestyle underground. Also, the mixed infection of nematodes in field crops complicates the need for more accurate diagnostic and quantification technologies. To address this challenge, we developed and optimized a novel duplex droplet digital PCR (ddPCR) method, using primer/probe sets targeting M. incognita and M. enterolobii, to simultaneously identify and quantify both species within a single assay. The innovative ddPCR diagnostic demonstrated excellent performance in terms of sensitivity, precision and reproductivity when quantifying the eggs and soil samples containing juveniles of both species. Moreover, the application of the duplex ddPCR method enables the monitoring of population dynamics of M. incognita and M. enterolobii under competitive environmental conditions. Our results indicated that the reproduction factor of M. incognita possibly inhibited when in mixed populations of M. enterolobii. In this study, we first applied duplex ddPCR technique for differentiating mixed infections of M. incognita and M. enterolobii, offering a valuable tool for species detection and quantification. It enables the monitoring of population dynamics for both species, which is crucial for providing theoretical guidance for the implementation of timely and effective control measures. © 2024 Society of Chemical Industry.

Strategy to develop and validate digital droplet PCR methods for global antimicrobial resistance wastewater surveillance.

According to World Health Organization (WHO), antimicrobial resistance (AMR) is currently one of the world's top 10 health threats, causing infections to become difficult or impossible to treat, increasing the risk of disease spread, severe illness, disability, and death. Accurate surveillance is a key component in the fight against AMR. Wastewater is progressively becoming a new player in AMR surveillance, with the promise of a cost-effective real-time tracking of global AMR profiles in specific regions. One of the most useful analytical methods for wastewater surveillance is currently based on real-time PCR (qPCR) and digital droplet PCR (ddPCR) technologies. As stated in the EU Wastewater Treatment Directive proposal, methodological standardization, including a workflow for method development and validation, will play a crucial role in global monitoring of AMR in wastewater. However, according to our knowledge, there are currently no qPCR and ddPCR methods for AMR surveillance available that have been validated according to international standard performance criteria. Therefore, this study proposes a workflow for the development and validation of PCR-based methods for a harmonized and global AMR surveillance, including the construction of specific sequence databases and microbial collections for an efficient method development and method specificity evaluation. Following this strategy, we have developed and validated four duplex ddPCR methods responding to international standard performance criteria, focusing on seven AMR genes (ARG's), including extended spectrum beta-lactam (blaCTX-M), carbapenem (blaKPC-2/3), tetracycline (tet(M)), erythromycin (erm(B)), vancomycin (vanA), sulfonamide (sul2), and aminoglycoside (aac(3)-IV), as well as one indicator of antibiotic (multi-) resistance and horizontal gene transfer, named the class I integron (intl1). The performance of these ddPCR methods was successfully assessed for their specificity, as no false-positive and false-negative results were observed. These ddPCR methods were also considered to be highly sensitive as showing a limit of detection below 25 copies of the targets. In addition, their applicability was confirmed using 14 wastewater samples collected from two Belgian water resource recovery facilities. The proposed study represents therefore a step forward to reinforce method harmonization in the context of the global AMR surveillance in wastewater. PRACTITIONER POINTS: In the context of wastewater surveillance, no PCR-based methods for global AMR monitoring are currently validated according to international standards. Consequently, we propose a workflow to develop and validate PCR-based methods for a harmonized and global AMR surveillance. This workflow resulted here in four duplex ddPCR methods targeting seven ARGs and one general indicator for mobilizable resistance genes. The applicability of these validated ddPCR methods was confirmed on 14 wastewater samples from two Belgian water resource recovery facilities.

Discovery unbalanced DNA mixtures and evaluation mixing ratio via a droplet digital PCR method.

Small amounts of DNA from a perpetrator collected during crime-scene investigations can be masked by large amounts of DNA from the victim. These samples can provide important information for the perpetrator's conviction. Short tandem repeat (STR) detection system is not sensitive enough to detect trace amounts of minor components in unbalanced mixed DNA. We developed a system using droplet digital polymerase chain reaction (ddPCR) capable of discovering trace components and accurately determining the ratio of mixed DNA in extremely unbalanced mixtures. The non-recombining regions of the X chromosome and Y chromosome were quantified in the DNA of male and female mixtures using duplex ddPCR. Absolute quantification of low-abundance portions of trace samples and unbalanced mixtures was done using different mixing ratios. The ddPCR system could be used to detect low-abundance samples with < 5 copies of DNA components in an extremely unbalanced mixture at a mixing ratio of 10000:1. The high sensitivity and specificity of the system could identify the mixing ratio of mixed DNA accurately. A ddPCR system was developed for evaluation of mixed samples of male DNA and female DNA. Our system could detect DNA quantities as low as 5 copies in extremely unbalanced mixed samples with good specificity and applicability. This method could assist forensic investigators in avoiding the omission of important physical evidence, and evaluating the ratio of mixed male/female trace samples.

Duplex Droplet Digital PCR Assay for Quantification of Hepatitis E Virus in Food.

Hepatitis E virus (HEV) represents an emerging risk in industrialized countries where the consumption of contaminated food plays a pivotal role. Quantitative real-time RT-PCR (RT-qPCR) is one of the most suitable methods for the detection and quantification of viruses in food. Nevertheless, quantification using RT-qPCR has limitations. Droplet digital PCR (ddPCR) provides the precise quantification of nucleic acids without the need for a standard curve and a reduction in the effect on virus quantification due to the presence of inhibitors. The objectives of the present work were (i) to develop a method for the absolute quantification of HEV in swine tissues based on ddPCR technology and provide internal process control for recovery assessment and (ii) to evaluate the performance of the method by analyzing a selection of naturally contaminated wild boar muscle samples previously tested using RT-qPCR. The method was optimized using a set of in vitro synthesized HEV RNA and quantified dsDNA. The limit of detection of the developed ddPCR assay was 0.34 genome copies/µL. The analysis of the wild boar samples confirmed the validity of the ddPCR assay. The duplex ddPCR method showed no reduction in efficiency compared to individual assays. The method developed in the present study could represent a sensitive assay for the detection and absolute quantification of HEV RNA in food samples with the advantage of presenting the co-amplification of internal process control.

Development of a droplet digital polymerase chain reaction assay for the sensitive detection of total and integrated HIV-1 DNA.

Total human immunodeficiency virus (HIV) DNA and integrated HIV DNA are widely used markers of HIV persistence. Droplet digital polymerase chain reaction (ddPCR) can be used for absolute quantification without needing a standard curve. Here, we developed duplex ddPCR assays to detect and quantify total HIV DNA and integrated HIV DNA. The limit of detection, dynamic ranges, sensitivity, and reproducibility were evaluated by plasmid constructs containing both the HIV long terminal repeat (LTR) and human CD3 gene (for total HIV DNA) and ACH-2 cells (for integrated HIV DNA). Forty-two cases on stable suppressive antiretroviral therapy (ART) were assayed in total HIV DNA and integrated HIV DNA. Correlation coefficient analysis was performed on the data related to DNA copies and cluster of differentiation 4 positive (CD4 + ) T-cell counts, CD8 + T-cell counts and CD4/CD8 T-cell ratio, respectively. The assay linear dynamic range and lower limit of detection (LLOD) were also assessed. The assay could detect the presence of HIV-1 copies 100% at concentrations of 6.3 copies/reaction, and the estimated LLOD of the ddPCR assay was 4.4 HIV DNA copies/reaction (95% confidence intervals [CI]: 3.6-6.5 copies/reaction) with linearity over a 5-log 10 -unit range in total HIV DNA assay. For the integrated HIV DNA assay, the LLOD was 8.0 copies/reaction (95% CI: 5.8-16.6 copies/reaction) with linearity over a 3-log 10 -unit range. Total HIV DNA in CD4 + T cells was positively associated with integrated HIV DNA ( r = 0.76, P <0.0001). Meanwhile, both total HIV DNA and integrated HIV DNA in CD4 + T cells were inversely correlated with the ratio of CD4/CD8 but positively correlated with the CD8 + T-cell counts. This ddPCR assay can quantify total HIV DNA and integrated HIV DNA efficiently with robustness and sensitivity. It can be readily adapted for measuring HIV DNA with non-B clades, and it could be beneficial for testing in clinical trials.

Development of a duplex droplet digital PCR assay for the detection of Burkholderia cepacia complex and Stenotrophomonas maltophilia in bloodstream infections.

Burkholderia cepacia complex (BCC) and Stenotrophomonas maltophilia are nosocomial pathogens that cause various infections and exhibit high resistance to multiple antimicrobial agents. In this study, we aimed to develop a duplex droplet digital PCR (ddPCR) assay for detecting BCC and S. maltophilia in bloodstream infections. We optimized the experimental conditions by setting the annealing temperature to 51°C and determining the optimal concentrations of primers and probes, as well as the thermal cycle numbers. The feasibility of the duplex ddPCR reaction system with the optimal conditions was established and verified through parallel reactions with reference strains of BCC and S. maltophilia. The specificity of the assay, tested with 33 reference strains, was found to be 100%. The duplex ddPCR assay demonstrated good repeatability and could detect as low as 5.35 copies/reaction of BCC and 7.67 copies/reaction of S. maltophilia. This level of sensitivity was consistent in the simulated blood and blood bottle samples. We compared nucleic acid extraction methods and found that the Chelex-100 boiling method and kit extraction method exhibited similar detection sensitivity, suggesting the potential application of the Chelex-100 boiling method in the ddPCR assay. In the clinical samples, the duplex ddPCR assay accurately detected BCC and S. maltophilia in 58 cases. In conclusion, our study successfully developed a duplex ddPCR assay that provides accurate and convenient detection of BCC and S. maltophilia in bloodstream infections.IMPORTANCEBurkholderia cepacia complex (BCC) and Stenotrophomonas maltophilia are implicated in a wide range of infections, including bloodstream infections (BSIs), pneumonia, and meningitis, and often exhibit high intrinsic resistance to multiple antimicrobial agents, limiting therapeutic options. The gold standard for diagnosing bloodstream infections remains blood culture. However, current blood culture detection and positivity rates do not meet the "rapid diagnosis" required for the diagnosis and treatment of critically ill patients with BSIs. The digital droplet PCR (ddPCR) method is a potentially more powerful tool in the diagnosis of BSIs compared to other molecular methods due to its greater sensitivity, specificity, accuracy, and reproducibility. In this study, a duplex ddPCR assay for the detection of BCC and S. maltophilia in BSIs was developed.

Multiplex ddPCR: A Promising Diagnostic Assay for Early Detection and Drug Monitoring in Bovine Theileriosis

Accurate quantification based on nucleic acid amplification is necessary to avoid the spread of pathogens, making early diagnosis essential. Droplet digital PCR (ddPCR) stands out for absolute parasite quantification because it combines microfluidics with the TaqMan test. This helps deliver maximum accuracy without needing a reference curve. This study assessed the efficacy of ddPCR as a detection tool for the bovine theileriosis (BT) caused by Theileria parasites. We developed and validated a duplex ddPCR method that detects and quantifies the Theileria genus (18S rRNA) and identifies clinically significant Theileria annulata parasites (TaSP) in experimental and clinical samples. ddPCR was shown to be as effective as qPCR throughout a 10-fold sample dilution range. However, ddPCR was more sensitive than qPCR at lower parasite DNA concentrations and reliably assessed up to 8.5 copies/µL of the TaSP gene in the infected DNA (0.01 ng) samples. The ddPCR was very accurate and reproducible, and it could follow therapeutic success in clinical cases of theileriosis. In conclusion, our ddPCR assays were highly sensitive and precise, providing a valuable resource for the study of absolute parasite quantification, drug treatment monitoring, epidemiological research, large-scale screening, and the identification of asymptomatic parasite reservoirs in the pursuit of BT eradication.

Genome concentration, characterization, and integrity analysis of recombinant adeno-associated viral vectors using droplet digital PCR.

Precise, reproducible characterization of AAV is critical for comparing preclinical results between laboratories and determining a safe and effective clinical dose for gene therapy applications. In this study, we systematically evaluated numerous parameters to produce a simple and robust ddPCR protocol for AAV characterization. The protocol uses a low ionic strength buffer containing Pluronic-F68 and polyadenylic acid to dilute the AAV into the ddPCR concentration range and a 10-minute thermal capsid lysis prior to assembling ddPCR reactions containing MspI. A critical finding is that the buffer composition affected the ITR concentration of AAV but not the ITR concentration of a double stranded plasmid, which has implications when using a theoretical, stoichiometric conversion factor to obtain the titer based on the ITR concentration. Using this protocol, a more comprehensive analysis of an AAV vector formulation was demonstrated with multiple ddPCR assays distributed throughout the AAV vector genome. These assays amplify the ITR, regulatory elements, and eGFP transgene to provide a more confident estimate of the vector genome concentration and a high-resolution characterization of the vector genome identity. Additionally, we compared two methods of genome integrity analysis for three control sample types at eight different concentrations for each sample. The genome integrity was independent of sample concentration and the expected values were obtained when integrity was determined based on the excess number of positive droplets relative to the number of double positive droplets expected by chance co-encapsulation of two DNA targets. The genome integrity was highly variable and produced unexpected values when the double positive droplet percentage was used to calculate the genome integrity. A protocol using a one-minute thermal capsid lysis prior to assembling ddPCR reactions lacking a restriction enzyme used the non-ITR assays in a duplex ddPCR milepost experiment to determine the genome integrity using linkage analysis.

Application of Duplex Droplet Digital PCR Detection of miR-888 and miR-891a in Semen Identification.

To establish a system for simultaneous detection of miR-888 and miR-891a by droplet digital PCR (ddPCR), and to evaluate its application value in semen identification. The hydrolysis probes with different fluorescence modified reporter groups were designed to realize the detection of miR-888 and miR-891a by duplex ddPCR. A total of 75 samples of 5 body fluids (including peripheral blood, menstrual blood, semen, saliva and vaginal secretion) were detected. The difference analysis was conducted by Mann-Whitney U test. The semen differentiation ability of miR-888 and miR-891a was evaluated by ROC curve analysis and the optimal cut-off value was obtained. There was no significant difference between the dual-plex assay and the single assay in this system. The detection sensitivity was up to 0.1 ng total RNA, and the intra- and inter-batch coefficients of variation were less than 15%. The expression levels of miR-888 and miR-891a detected by duplex ddPCR in semen were both higher than those in other body fluids. ROC curve analysis showed that the AUC of miR-888 was 0.976, the optimal cut-off value was 2.250 copies/μL, and the discrimination accuracy was 97.33%; the AUC of miR-891a was 1.000, the optimal cut-off value was 1.100 copies/μL, and the discrimination accuracy was 100%. In this study, a method for detection of miR-888 and miR-891a by duplex ddPCR was successfully established. The system has good stability and repeatability and can be used for semen identification. Both miR-888 and miR-891a have high ability to identify semen, and the discrimination accuracy of miR-891a is higher.

Quantitative detection of Vibrio parahaemolyticus in aquatic products by duplex droplet digital PCR combined with propidium monoazide

Based on the combination of droplet digital PCR (ddPCR) and propidium monoazide (PMA), a new method called PMA-duplex ddPCR was developed for the quantitative determination of viable Vibrio parahaemolyticus in aquatic products. Specific primers and probes targeting a new gene, vp0488, coding for putative DNA polymerase III beta chain, which was self-screened by this laboratory, and a traditional target gene, thermolabile hemolysin (tlh), were designed and verified. The duplex ddPCR system and conditions and PMA treatment conditions were optimized. The limit of detection (LOD) for PMA-duplex ddPCR was evaluated and applied to detect V. parahaemolyticus in artificially contaminated samples and actual samples. The results indicate that the optimal duplex ddPCR annealing temperature was 60 °C, the optimal number of amplification cycles was 40, and the optimal ratio of primer to probe was 1:0.5. The optimal PMA treatment conditions were as follows: incubation time 5 min, concentration 30 μmol/L, and exposure time 15 min. The LOD of PMA-duplex ddPCR (7.32 × 101 CFU/mL) was lower than that of duplex quantitative PCR (qPCR) (7.32 × 102 CFU/mL). The lowest detectable V. parahaemolyticus in oysters was 8.15 × 101 CFU/g without any pre-enrichment. The method established in this study has the advantages of good specificity, high sensitivity and accuracy, which provides an effective method for the detection of viable Vibrio parahaemolyticus in aquatic foods.

Validation of droplet digital PCR for cytokeratin 19 mRNA detection in canine peripheral blood and mammary gland

In humans, peripheral blood cytokeratin 19 (CK19) mRNA-positive circulating tumor cells (CTCs) was utilized to identify early-stage breast cancer patients with micrometastatic disease who are at risk for disease progression and monitor treatment response in patients with advanced disease. To our knowledge, there has been little research regarding CK19 in canine mammary tumors (CMTs) using molecular methods. A droplet digital PCR (ddPCR) is proposed as a precise and sensitive quantification of nucleic acid targets. Hence, this study aimed to validate a newly designed assay for CK19 detection in canine blood and mammary tissue, along with the reference gene HPRT, by ddPCR. All primers and probes showed a precise match with the exon region of target genes. The assay exhibited PCR efficacy of 90.4% and 91.0% for CK19 and HPRT amplifications with linearity, respectively. The annealing temperature (Ta) for duplex ddPCR was 55 °C, providing the highest concentrations of both genes tested by the synthetic plasmid DNA. The limit of detection (LOD) of CK19 and HPRT were 2.16 ± 1.27 and 2.44 ± 1.31 copies/µL, respectively. Finally, the ddPCR assay was validated with canine peripheral blood, non-neoplastic mammary tissues and spiked samples. Our findings provide a new platform for CK19 studies in CMT diagnosis through blood and mammary tissues.

Development and Application of a Duplex Droplet Digital Polymerase Chain Reaction Assay for Detection and Differentiation of EP402R-Deleted and Wild-Type African Swine Fever Virus.

African swine fever (ASF) is a highly fatal porcine disease caused by the African swine fever virus (ASFV), and resulting in huge economic losses across the globe. ASF has been raging in China for 3 years, and recently EP402R-deleted ASFV strains emerged, showing sub-acute or chronic symptoms in pigs and providing novel difficulties to monitor and control the disease as EP402R-deleted strains possess no hemadsorption (HAD) ability. In addition, the gene deletion virus with low viral load is prone to results retest or false negative due to the high cycle threshold (Ct) value under the current real-time polymerase chain reaction (PCR) detection method. Thus, a new method is needed to detect and distinguish wild strains and gene-deleted viruses. In this study, a duplex droplet digital polymerase chain reaction (ddPCR) assay based on the ASFV B646L and EP402R genes was established and showed good linearity (R2 > 0.99). The limit of detection for duplex ddPCR was 52 copies per reaction and 8.6 copies per reaction for B646L and EP402R, respectively. No cross-reaction with other porcine viruses [classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV), porcine parvovirus (PPV), Japanese encephalitis virus (JEV), and porcine circovirus type 2 (PCV2)] was identified by this assay. In addition, 44 ASFV-suspicious clinical samples as well as EP402R-deleted ASFV were tested in parallel by duplex real-time PCR and ddPCR, indicative of a higher sensitivity which belonged to the duplex ddPCR assay. In summary, this is the first time that duplex ddPCR assay has been successfully developed to provide an efficient method to detect and differentiate ASFV wild-type and gene-deleted strains.

Development of genetically modified maize (&amp;lt;italic&amp;gt;Zea mays &amp;lt;/italic&amp;gt;L.) NK603 matrix reference materials

<p id="C3">The implementation of genetically modified organisms (GMO) safety management and labeling system requires standardized testing methods and reference materials (RMs). Furthermore, the RMs are the guarantee of obtaining accurate, reliable and comparable testing results. At present, genetically modified (GM) maize (<italic>Zea mays</italic> L.) NK603 has been approved to be imported as processing raw material in China, and its safety supervision urgently needs to prepare RMs. In this study, the heterozygous seeds of GM maize NK603 and the non-GM maize counterpart after identification were well milled and then gravimetrically mixed to prepare three levels of powder RMs according to the mass fractions of 60.0, 100.0, and 1000 mg g^-1. The homogeneity and stability test by duplex ddPCR method showed that the RMs of GM maize NK603 were homogenous and stable for not less than six months. The property values were collaboratively characterized by eight laboratories using duplex ddPCR method. The certified values together with their expanded uncertainties were (2.75±0.26)%, (4.68±0.39)%, and (51.8±3.7)%, respectively. The minimum sample intake was determined to be 100 mg. This batch of RMs can be used for the qualitative and quantitative detection of NK603 event in food and feed, as well as for the evaluation of GM maize NK603-specific assays and laboratory quality control.

A duplex droplet digital PCR assay for Salmonella and Shigella and its application in diarrheal and non-diarrheal samples

ObjectivesTo evaluate a duplex droplet digital polymerase chain reaction (ddPCR) assay targeting Salmonella fimY and Shigella ipaH genes. MethodsThe linear range, precision, analytical sensitivity, and analytical specificity of the ddPCR assay were analyzed. The ddPCR assay was compared with quantitative real-time PCR (qPCR) using 362 stool samples from 187 children with mild diarrhea and 175 children without diarrhea. ResultsThe duplex ddPCR assay showed good linearity in the range of 5.3 × 100 to 1.24 × 105 copies/reaction for Salmonella and 1.9 × 100 to 1.84 × 105 copies/reaction for Shigella. When analyzed with spiked stool samples, the limit of detection and limit of quantification were 550 and 5500 colony-forming units per mL of stool sample for Shigella, respectively, whereas both were 1.0 × 104 colony-forming units per mL of stool sample for Salmonella. Among 362 stool samples, more samples were detected as positive by ddPCR than by qPCR. Salmonella load was significantly higher in diarrheal samples than in non-diarrheal samples. Determined by receiver-operating characteristic analysis, the optimal cut-off value was 1.25 × 104 copies/mL of stool sample to distinguish between symptomatic and asymptomatic Salmonella infections. ConclusionSalmonella and Shigella prevalence may have been underestimated in the past.

A novel duplex droplet digital PCR assay for simultaneous authentication and quantification of Panax notoginseng and its adulterants

Food adulteration can result in unfair commercial trade and consumer distrust, therefore, a reliable and effective method for food qualitative and quantitative analysis is necessary. In the present work, we developed a highly sensitive and precise approach based on duplex droplet digital PCR (ddPCR) for the identification and quantification of Panax notoginseng (P. notoginseng) powder and its adulterants. The detection targeted single copy nuclear genes, the limits of detection (LODs) for rice and soybean were both 0.1%, and the limits of quantifications (LOQs) were both 0.5%. The relationship between species proportions and DNA copy number was established for rice, soybean and P. notoginseng and the regression equation all exhibited good linearity. To further demonstrate the accuracy of the developed method, samples with known concentrations were tested. In general, the developed duplex ddPCR assays is useful for addressing adulteration issues in health food material powder from P. notoginseng.

Increasing the Efficiency of Canola and Soybean GMO Detection and Quantification Using Multiplex Droplet Digital PCR.

Simple SummaryDigital PCR (dPCR) technology has been used for absolute quantification of genetically modified (GM) events. Duplex dPCR consisting of a target gene and a reference gene is mostly used for absolute quantification of GM events. We investigated the feasibility of absolute quantification of two, three, and four GM canola and soybean events at the same time using the QX200 Droplet Digital PCR (ddPCR) system. Adjustments of the probe concentrations and labels for some of the assays were needed for successful multiplex ddPCR. Absolute quantification of GM canola and soybean events was achieved for duplex, triplex, and tetraplex ddPCR at 0.1%, 1%, and 5% concentrations.The number of genetically modified (GM) events for canola, maize, and soybean has been steadily increasing. Real-time PCR is widely used for the detection and quantification of individual GM events. Digital PCR (dPCR) has also been used for absolute quantification of GM events. A duplex dPCR assay consisting of one reference gene and one GM event has been carried out in most cases. The detection of more than one GM event in a single assay will increase the efficiency of dPCR. The feasibility of detection and quantification of two, three, and four GM canola and soybean events at the same time was investigated at 0.1%, 1%, and 5% levels using the QX200 Droplet Digital PCR (ddPCR) system. The reference gene assay was carried out on the same plate but in different wells. For some of the assays, optimization of the probe concentrations and labels was needed for successful ddPCR. Results close to the expected result were achieved for duplex, triplex, and tetraplex ddPCR assays for GM canola events. Similar ddPCR results were also achieved for some GM soybean events with some exceptions. Overall, absolute quantification of up to four GM events at the same time improves the efficiency of GM detection.

Droplet digital polymerase chain reaction (ddPCR) for rapid screening of adulterants in honey: A case study on acacia honey adulterated with canola honey

In this study, droplet digital polymerase chain reaction (ddPCR) was used to detect adulteration of acacia honey with canola honey. DNA extraction from pollen in acacia honey and canola honey was performed using four different pollen treatment methods, including, enzymatic hydrolysis, freeze-thaw, mechanical, and ultrasonic. The mechanical extraction method was the most preferred method of DNA extraction from honey. A duplex ddPCR method developed based on the specific target gene in acacia and canola was performed. The detection results showed that the established duplex ddPCR had a high detection sensitivity, detecting up to 1% adulteration. The accuracy of duplex ddPCR was consistent with the traditional melissopalynologic method. All results indicated that ddPCR was a powerful method for assessing the authenticity and quality of honey. • A pollen treatment method for extraction DNA from honey was proposed. • A duplex ddPCR based on the specific target gene in acacia and canola was developed. • The duplex ddPCR method had a high detection sensitivity, detecting up to 1% adulteration. • The method was applied to assess real samples to demonstrate the practicability for adulteration detection.

Duplex droplet digital PCR (ddPCR) method for the quantification of common wheat (Triticum aestivum) in spelt (Triticum spelta)

A duplex droplet digital PCR (ddPCR) is described for the detection and quantification of contaminations by common wheat (Triticum aestivum) in food products made from spelt (Triticum spelta). A single nucleotide polymorphism (SNP) in the locus Q, as well as a short sequence of the γ-gliadin gene of the wheat genome are exploited for the development of our duplex ddPCR method. Minimal variations of DNA sequences causing different probe hydrolysis efficiencies during PCR amplification can be detected and evaluated in ddPCR. By utilizing the SNP in the locus Q, all 84 tested spelt cultivars could unambiguously discriminated from common wheat cultivars. Discrimination using the γ-gliadin gene sequence could be achieved for 10 of 84 spelt cultivars. Additionally, we could show that impurities caused by common wheat can also be detected in einkorn wheat (T. monococcum) and in emmer (T. dicoccon).

Development of species-specific primers and highly sensitive duplex ddPCR assay for the identification and detection of chili anthracnose

Development of species-specific primers and highly sensitive duplex ddPCR assay for the identification and detection of chili anthracnose

Event-specific PCR methods to quantify the genetically modified DBN9936 maize

The genetically modified (GM) maize DBN9936 that has been granted a biosafety certificate needs to be regulated in China. An event-specific PCR assay targeting the junction region between the left border of its T-DNA and the flanking genomic DNA (gDNA) was established to be able to specifically identify the DBN9936 event from other GM maize and non-GM maize. Both qPCR and duplex ddPCR achieved accurate measurement of DBN9936 content in blind matrix and gDNA samples with acceptable precision in terms of a relative standard deviation (RSD) of <25 % and a trueness in terms of relative bias of ±25 %. The quantitative values of the same sample did not show significant difference between qPCR and duplex ddPCR, whereas the duplex ddPCR showed advantage over qPCR in terms of precision. This study provided an event-specific PCR method for identification and quantification of DBN9936 on both a real-time PCR platform and a ddPCR platform.