EvaGreen Real-time PCR Research Articles

Development of a Melting Curve-Based Triple Eva Green Real-Time PCR Assay for Simultaneous Detection of Three Shrimp Pathogens.

Infections with Enterocytozoon hepatopenaei (EHP), infectious hypodermal and hematopoietic necrosis virus (IHHNV), and Decapod iridescent virus 1 (DIV1) pose significant challenges to the shrimp industry. Here, a melting curve-based triple real-time PCR assay based on the fluorescent dye Eva Green was established for the simultaneous detection of EHP, IHHNV, and DIV1. The assay showed high specificity, sensitivity, and reproducibility. A total of 190 clinical samples from Shandong, Jiangsu, Sichuan, Guangdong, and Hainan provinces in China were evaluated by the triple Eva Green real-time PCR assay. The positive rates of EHP, IHHNV, and DIV1 were 10.5%, 18.9%, and 44.2%, respectively. The samples were also evaluated by TaqMan qPCR assays for EHP, DIV1, and IHHNV, and the concordance rate was 100%. This illustrated that the newly developed triple Eva Green real-time PCR assay can provide an accurate method for the simultaneous detection of three shrimp pathogens.

Designing and Development of Simultaneous Detection of Neisseria meningitidis and Streptococcus pneumoniae based on EvaGreen Real-Time PCR

Background: Neisseria meningitidis and Streptococcus pneumoniae are serious causes of invasive infections associated with high mortality and morbidity worldwide, particularly meningitis. Efficient diagnostic strategies play a crucial role in the management of disease and the prevention of overtreatment. The low sensitivity and time-consuming nature of culture and gram stain methods have led to the demand for alternative methods in clinical laboratories. Objectives: This study aims to design and develop a rapid, sensitive, and cost-effective EvaGreen-based real-time PCR to simultaneously detect N. meningitidis and S. pneumoniae. Methods: We designed and evaluated an accurate, reliable, and inexpensive approach based on EvaGreen dye real-time PCR to simultaneously detect N. meningitidis and S. pneumoniae in a single tube from cerebrospinal fluid. Melting curve analysis was used to differentiate the amplicons of each pathogen. Analytical sensitivity and specificity of the assay were conducted by reference bacterial strains genomes. Besides, in order to clinical validation we used 53 positive CSF samples and 7 negative CSF samples. Results: Our assay demonstrated no amplification curve with non-target microorganisms indicating 100% analytical specificity. In the EvaGreen multiplex assay, the lower limit of detection (LLD) was nine copies/reaction for N. meningitidis and 13 copies/reaction for S. pneumoniae. The clinical validation of positive CSF samples revealed 100% sensitivity and no false positives. The reproducibility and repeatability of tested replicates indicated low intra-assay and inter-assay CVs of less than 1.5%. Conclusions: EvaGreen-based multiplex real-time PCR offers a rapid, affordable, and appropriate diagnostic tool to identify the main cause of bacterial meningitis.

An Eva Green Real-Time PCR Assay for Porcine DNA Analysis in Raw and Processed Foods

Abstract The prevalence of the inclusion or substitution of porcine and its derivatives in foods by imprudent food manufacturers accentuated the need for a sensitive and specific method to detect porcine DNA. This work aimed to develop and validate an EvaGreen dye-based quantitative real-time (qPCR) assay for porcine DNA analysis. The primer set used in this work targets a 95-bp fragment of the porcine cytochrome b gene. Application of the assay to dilutions of porcine genomic DNA showed that the assay is sensitive down to 10 pg/μL of porcine DNA and the detection limit, under binary admixture conditions, was 0.001%. A correlation coefficient (R2) of 0.998 and PCR efficiency of 101.6% over the dynamic range from 10 to 10000 pg/μL indicated that the assay has high linearity and efficiency. The method’s specificity towards porcine was revealed by the amplification profile, which only displayed an amplification signal for porcine DNA. These findings suggest that the developed assay is sensitive, specific, and rapid. In conclusion, the assay can be used as an alternative method to detect and quantify porcine DNA in raw and processed food products.

Development of an EvaGreen-based real-time PCR assay for detection of Aleutian mink disease virus

The objective of this study was to develop a rapid, sensitive and specific EvaGreen (EG)-based real-time PCR assay capable of detecting Aleutian mink disease virus (AMDV) and to evaluate the reliability of the assay for analysis of blood or tissue samples. For this assay, a pair of primers was designed based on a nonstructural protein (NS)-encoding gene of AMDV, and the identity of PCR products was identified based on a melting temperature of 82.8°C. The EG-based real-time PCR assay did not detect canine distemper virus or mink enteritis virus, and the assay could be used to detect Chinese and American AMDV strains, in contrast to a commercial TaqMan kit that could only detect American AMDV strains. The amplification efficiencies of the EG assay were 104.8% for the Chinese strain and 94.4% for the American strain, and the detection limit was 1 copy/μL of AMDV plasmid or 3 pg/μL of viral DNA (Chinese strain). The intra- and inter-assay variation coefficients of melting temperature were all lower than 0.15%, confirming the high reproducibility of the assay. Forty-five clinical blood samples were simultaneously analyzed using the EG real-time PCR, TaqMan kit and conventional PCR, and the detection rates were 91.1%, 0.0% and 86.7%, respectively. Serum samples were also collected from the corresponding blood samples and tested using the counterimmunoelectrophoresis (CIEP) assay, where positive samples accounted for 24.4% of the 45 samples. In conclusion, EG-based real-time PCR is a rapid, sensitive, universal assay that can be effectively utilized as a reliable and specific tool for detection and quantitation of AMDV.

Simultaneous detection of five pig viruses associated with enteric disease in pigs using EvaGreen real-time PCR combined with melting curve analysis

In recent years, a series of porcine diarrhea viruses such as porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), rotaviruses of group A (RVA), rotaviruses of group C (RVC), and porcine circovirus 2 (PCV2) caused enormous economic losses all over the world. While any of these viruses is capable to cause disease alone, there is often concurrent infection with more than one virus on pig farms. In this study, a multiplex real-time PCR method based on EvaGreen fluorescent dye and melting curve analysis was established to simultaneously detect these five viruses in a single closed tube. Five distinct melt peaks were obtained with different melting temperature (Tm) value corresponding to each of the five viruses. This method was highly sensitive to detect and distinguish TGEV, RVA, RVC, PEDV and PCV2 with the limits of detection ranging from 5 to 50 copies/μL. The intra-assay and inter-assay reproducibility were good with coefficient of variation of Tm and cycle threshold values less than 0.32% and 2.86%, respectively. Testing of 90 field samples by the single and multiplex real-time PCR assays demonstrated a concordance of 91.1%. Thus, the EvaGreen multiplex real-time PCR is a rapid, sensitive and low-cost diagnostic tool for differential detection and routine surveillance of TGEV, RVA, RVC, PEDV and PCV2 in pigs.

EvaGreen-based real-time PCR assay for sensitive detection of salmonid alphavirus

Salmonid alphaviruses (SAVs), which include the etiological agents of salmon pancreas disease (PD) and sleeping disease (SD), are significant viral pathogens of European salmonid aquaculture, resulting in substantial economic losses to the salmonid-farming industry. Even though many countries including China have not reported the presence of SAV infections, these countries may be seriously threatened by these diseases as the salmon fish import trade increases. Thus, it is indeed necessary to develop efficient detection methods for the diagnosis and prevention of SAV infection. Real-time PCR assays have been increasingly used in viral detection, and in many cases scientists prefer dye-based real-time PCR assays for their high sensitivity and low cost. In this study, we developed a novel, sensitive, low-cost detection method, EvaGreen-based real-time PCR assay for the detection of SAV. This assay exhibited high specificity for SAV1, SAV2, and SAV5 and was able to detect SAV at concentrations as low as 1.5 × 101 copies, making them more sensitive than the approved conventional RT-PCR method (detection limit, 1.5 × 106 copies). Assessment of infected fish samples showed that the sensitivity of EvaGreen-based assay was higher than previously developed SYBR Green assay (227 assay). Thus, we report that the EvaGreen real-time PCR assays is an economical alternative diagnostic method for the rapid detection of SAV1, SAV2, and SAV5 infection, providing improved technical support for the clinical diagnosis and epidemiological investigation of SAV.

Development of Simple Multiplex Real-Time PCR Assays for Foodborne Pathogens Detection and Identification On Lightcycler

Abstract Most acute intestinal diseases are caused by food-borne pathogens. A fast and simple real-time PCR-based procedure for simultaneous detection of food contamination by any of the five food-borne pathogens: Campylobacter jejuni, Mycobacterium bovis, Enterobacter sakazaki, Shigella boydii, Clostridium perfrigens using multiplex EvaGreen real-time PCR for LightCycler was developed and evaluated. Real-time qPCR showed excellent sensitivity. Tm calling and Melting Curve Genotyping (MCG) were used for analysis of PCR product melting curves. The Melting Curve Genotyping option showed good performance for discrimination of positive samples containing DNA of single pathogen or pathogen mixtures from negative samples.

EvaGreen real-time PCR to determine horse meat adulteration in processed foods

The aim of this work was the development of qualitative and quantitative methods for the detection of horse meat adulteration in processed foods. The design of new primers targeting the cytb gene of Equus caballus enabled proposing two types of assays: species-specific PCR and real-time quantitative PCR with EvaGreen dye. The assays demonstrated high sensitivity and specificity, down to 0.0001% (w/w) of horse meat in beef mixtures and 0.1 pg of horse DNA, in both raw and autoclaved meats. A normalised real-time PCR approach based on the ΔCt method was further proposed in the range of 25% down to 0.0001%, which was effectively validated and applied to a total of 67 processed foods. The estimated horse meat content (0.10%) in one sample suggests the occurrence of cross-contamination rather than adulteration.

Quantitative detection of pork meat by EvaGreen real-time PCR to assess the authenticity of processed meat products

The development of highly sensitive and quantitative tools to identify undeclared pork meat is very important to authenticate processed meat products and, particularly, in the case of Halal products. Quantitative approaches are crucial to distinguish deliberate adulterations from cross-contaminations. This study intended to develop and validate a novel specific and highly sensitive Evagreen real-time PCR system for pork meat quantification in processed meat products. A normalised assay based on the ΔCt method was successfully developed and optimised, allowing the detection and quantification of levels down to 0.0001% and 0.01% (w/w) of pork meat, respectively, in both raw and thermally processed. The method was effectively validated using blind meat mixtures, exhibiting adequate parameters of trueness, precision and repeatability. Its application to several commercial samples, including Halal and regular meat products, showed the presence of undeclared pork species in 54% of the analysed samples, with 40% of Halal products presenting traces of pork, therefore not in good agreement with their label statements.

Development of a single-tube duplex EvaGreen real-time PCR for the detection and identification of EHV-1 and EHV-4

The objective of this study was to develop a novel EvaGreen (EG) based real-time PCR technique for the simultaneous detection of Equine herpesvirus 1 (EHV-1) and Equine herpesvirus 4 (EHV-4) genomes from equine nasal swabs. Viral genomes were identified based on their specific melting temperatures (T m), which are 88.0 and 84.4°C for EHV-1 and EHV-4, respectively. The detection limitation of this method was 50 copies/μl or 0.15pg/μl for EHV-1 and 5 copies/μl or 2.5fg/μl for EHV-4. This assay was 50-1,000 times more sensitive than the SYBR Green (SG)-based assay using the same primer pairs and as sensitive as the TaqMan-MGB probe-based assay. The validity of the real-time PCR assays was confirmed by testing 13 clinical samples. When all results of the EG, SG, and TaqMan probe-based singleplex and duplex real-time PCRs were considered together, a total of 84.6% (11/13) horses and donkeys were positive for at least one virus. EHV-1 and EHV-4 coexisted in 81.8% (9/11) horses. Overall, we report that the EvaGreen duplex real-time PCR is an economical and alternative diagnostic method for the rapid differentiation of EHV-1 and EHV-4 in nasal swabs.

Advances in molecular surveillance of Clostridium difficile in Bulgaria

The increasing incidence of Clostridium difficile infection (CDI) in Bulgaria has indicated the need to implement better surveillance approaches. The aim of the present work was to improve the current surveillance of CDI in Bulgaria by introducing innovative methods for identification and typing. One hundred and twenty stool samples obtained from 108 patients were studied over 4 years from which 32 C. difficile isolates were obtained. An innovative duplex EvaGreen real-time PCR assay based on simultaneous detection of the gluD and tcdB genes was developed for rapid C. difficile identification. Four toxigenic profiles were distinguished by PCR: A(+)B(+)CDT(-) (53.1 %, 17/32), A(-)B(+)CDT(-) (28.1 %, 9/32), A(+)B(+)CDT(+) (9.4 %, 3/32) and A(-)B(-)CDT(-) (9.4 %, 3/32). PCR ribotyping and multilocus variable number of tandem repeat analysis (MLVA7) were used for molecular characterization of the isolates. In total, nine distinct ribotypes were confirmed and the most prevalent for Bulgarian hospitals was 017 followed by 014/020, together accounting for 44 % of all isolates. Eighteen per cent of the isolates (6/32) did not match any of the 25 reference ribotypes available in this study. Twenty-four MLVA7 genotypes were detected among the clinical C. difficile isolates, distributed as follows: five for 017 ribotype, two for 014/020, 001, 002, 012 and 046 each, and one each for ribotypes 023, 070 and 078. The correlation between the typing methods was significant and allowed the identification of several clonal complexes. These results suggest that most C. difficile cases in the eight Bulgarian hospitals studied were associated with isolates belonging to the outbreak ribotypes 017 and 014/20, which are widely distributed in Europe. The real-time PCR protocol for simultaneous detection of gluD and tcdB proved to be very effective and improved C. difficile identification and confirmation of clinical C. difficile isolates.

EvaGreen Real-time PCR protocol for specific ‘Candidatus Phytoplasma mali’ detection and quantification in insects

In this paper the validation and implementation of a Real-time PCR protocol based on ribosomal protein genes has been carried out for sensitive and specific quantification of ‘Candidatus (Ca.) Phytoplasma mali’ (apple proliferation phytoplasma, APP) in insects. The method combines the use of EvaGreen® dye as chemistry detection system and the specific primer pair rpAP15f-mod/rpAP15r3, which amplifies a fragment of 238 bp of the ribosomal protein rplV (rpl22) gene of APP. Primers specificity was demonstrated by running in the same Real-time PCR ‘Ca. Phytoplasma mali’ samples with phytoplasmas belonging to the same group (16SrX) as ‘Ca. Phytoplasma pyri’ and ‘Ca. Phytoplasma prunorum’, and also phytoplasmas from different groups, as ‘Ca. Phytoplasma phoenicium’ (16SrIX) and Flavescence dorée phytoplasma (16SrV). ‘Ca. Phytoplasma mali’ titre in insects was quantified using a specific approach, which relates the concentration of the phytoplasma to insect 18S rDNA. Absolute quantification of APP and insect 18S rDNA were calculated using standard curves prepared from serial dilutions of plasmids containing rplV-rpsC and a portion of 18S rDNA genes, respectively. APP titre in insects was expressed as genome units (GU) of phytoplasma per picogram (pg) of individual insect 18S rDNA. ‘Ca. Phytoplasma mali’ concentration in examined samples (Cacopsylla melanoneura overwintered adults) ranged from 5.94 × 102 to 2.51 × 104 GU/pg of insect 18S rDNA. Repeatability and reproducibility of the method were also evaluated by calculation of the coefficient of variation (CV%) of GU of phytoplasma and pg of 18S rDNA fragment for both assays. CV less than 14% and 9% (for reproducibility test) and less than 10 and 11% (for repeatability test) were obtained for phytoplasma and insect qPCR assays, respectively. Sensitivity of the method was also evaluated, in comparison with conventional 16S rDNA-based nested-PCR procedure. The method described has been demonstrated reliable, sensitive and specific for the quantification of ‘Ca. Phytoplasma mali’ in insects. The possibility to study the trend of phytoplasma titre in the vectors will allow a deepen investigation on the epidemiology of the disease.

Eva Green Real-time PCR used to detect celery as an allergen in food

EvaGreen® Real-Time PCR method has been used for celery(Apium graveolens) allergen detection. A primer designed in mannitol dehydrogenase gene region has been used for specific celery identification in sample. The results show possibility to create calibration curve using artificially adulterated samples. The increasing variability between parallel calibration of celery samples has been observed from 0.1 % to 100%. Detection limit has been set to value 0.1% in celery representing 1000 ppm. Fluorescent signal has been presented even in samples with lower percentage addition of celery but these samples have been excluded according to unspecific

Detection of olive oil using the Evagreen real-time PCR method

A sensitive real-time PCR method using the novel fluorescence stain Evagreen was established for detection of olive oil. First, a comparative study was made of two different methods for the recovery of high quality DNA from oil samples. Thereby, the Promega wizard DPSF kit proved to be the most suitable for recovery of DNA from oil samples. Second, an olive-specific pair of primers was designed based on the sequence of an olive plasma intrinsic protein cDNA sequence. Experiments with 13 samples including olive, soybean, sesame, sunflower seed, pumpkinseed, walnut, rapeseed, rice, peanut, maize, pig, chicken and fish confirmed that this pair of primers is highly specific for olive. Additional experiments indicated that the absolute detection limit of our test is 0.07 ng olive DNA and that 0.5% olive DNA can be detected against background of 2 ng/μl sesame DNA.