TaqMan Minor Groove Binder Probes Research Articles

Molecular genotyping of Babesia caballi

Babesia caballi is an intra-erythrocytic parasite causing equine piroplasmosis. Three B. caballi genotypes (A, B, and C) have been identified based on the 18S rRNA and rhoptry-associated protein (rap-1) gene sequences. These variant parasite genotypes compromise the diagnostic utility of the WOAH-recommended serological assays in declaring horses free of equine piroplasmosis. Although a gene encoding a spherical body protein 4 (sbp4) has recently been identified as a potential antigen for the serological detection of B. caballi, the ability of this antigen to detect the different geographical strains has not been determined. The molecular distinction between variant B. caballi genotypes is limited and therefore we developed molecular typing assays for the rapid detection and quantification of distinct parasite genotypes. Field samples were screened for the presence of B. caballi using an established multiplex equine piroplasmosis qPCR assay. In this study, B. caballi genotype A was not detected in any field samples screened. However, phylogenetic analysis of the amplified sbp4 and 18S rRNA genes confirmed the phylogenetic groupings of the South African isolates into either B. caballi genotypes B or C. A multiple sequence alignment of the sbp4 gene sequences obtained in this study together with the published sbp4 sequences representing B. caballi genotype A, were used to identify conserved regions within the gene to design three primer pairs and three genotype-specific TaqMan minor-groove binder (MGB™) probes. The qPCR assays were shown to be specific and efficient in the detection and differentiation between B. caballi genotypes A, B, and C and could be used as a diagnostic assay to prevent the unintentional spread of variant B. caballi genotypes globally.

Development of a Sensitive, Easy and High-Throughput Compliant Protocol for Maize and Soybean DNA Extraction and Quantitation Using a Plant-Specific Universal Taqman Minor Groove Binder Probe.

We report the optimization of a high-throughput, compliant DNA extraction method that uses standard format 96-well plates and a commercial automated DNA purification system (ABI PRISM® 6100 Nucleic Acid PrepStation). The procedure was set up for maize and soybean, the most common GMO crops and the main ingredients of several foodstuffs, and compared with an EU-validated CTAB-based method. Optimization of the DNA extraction was achieved by applying self-prepared buffers (for DNA extraction, binding, and washing) on the PrepStation loaded with proprietary glass-fiber-coated purification plates. Quantification of extracted DNA was performed by real-time PCR using previously reported endogenous soybean lectin and maize starch synthase genes and a novel plant-specific universal TaqMan MGB probe that targets the 18S rRNA multiple copy gene. Using serial dilutions of both maize and soybean genomic DNAs, we show low PCR sensitivity and efficiency for the official TransPrep DNA extraction protocol compared to the CTAB-based one. On the other hand, using serial dilutions of a standard reference plasmid containing a 137 bp sequence cloned from the 18S rRNA plant-specific ribosomal gene, we demonstrate the high PCR sensitivity and efficiency of the optimized DNA extraction protocol setup with self-prepared buffers. The limits of detection and quantification of the 18S rDNA reiteration were consistent with the calculated values, supporting the suitability of the DNA extraction procedure for high-throughput analyses of large populations and small amounts of tissue.

Development and validation of a PCR-RFLP / TaqMan MGB probe method for rapid sex identification of largemouth bass (Micropterus salmoides)

The largemouth bass (Micropterus salmoides) has become an important freshwater fish in China since it was intentionally introduced in the 1980 s, and the amount produced in aquaculture is increasing each year. Females are preferred because they grow faster than males and reach a larger body size. However, the physiological sex cannot be accurately determined from external morphology at the early growth stage, so it is crucial to develop sex-specific molecular markers. In this study, a single nucleotide polymorphism (SNP) marker was developed based on whole genome resequencing data obtained from 30 females and 30 males. This marker made it possible to positively identify male and female largemouth bass. Through Fst value screening, 123 SNP loci significantly associated with sex were obtained, and nearly 40% of them were located on chromosome 7 in a 3.44-Mb sex-linked region. A method of polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) combined with a TaqMan minor groove binder (MGB) probe was developed based on a sex-specific marker. Using this method, male and female fish in four different populations (n = 128) were identified with 100% accuracy. Compared with PCR-RFLP followed by electrophoretic separation of fragments, the PCR-RFLP–TaqMan MGB probe method reduced the time required to identify the sex of largemouth bass. The validation results also indicated that largemouth bass has an XX/XY sex-determination system. In conclusion, we have identified a sex-specific SNP marker for largemouth bass and developed a new technique to identify the sex of largemouth bass rapidly and accurately. These findings provide a scientific basis for further research on mono-sex breeding and the molecular mechanism of sex determination in largemouth bass.

Detection and quantification of Klebsiella pneumoniae in American bullfrog (Rana catesbeiana) by Taqman MGB probe real-time fluorescence quantitative PCR

Klebsiella pneumoniae is an important pathogen in American bullfrog farming. It causes serious diseases, resulting in significant losses. However, the spread and replication of K. pneumoniae in infected bullfrog tissues are poorly understood. This study aimed to investigate the variation in K. pneumoniae loads in the organs of infected bullfrogs to clarify the effect of K. pneumoniae replication. In this work, a TaqMan minor groove binder probe fluorescence real-time quantitative polymerase chain reaction (qPCR) assay was developed to rapidly detect and quantify K. pneumoniae.The detection limit of the qPCR method was as low as 2.9 copies/μL, which was 100 times more sensitive than that of the conventional PCR. In addition, comparison of K. pneumoniae in tissue mixing and mucus showed similar positive rates, K. pneumoniae in bullfrogs could be preliminarily detected and monitored by anal mucus detection under noninvasive conditions. The changes in bacterial load detected by qPCR in different tissues of bullfrogs infected with K. pneumoniae showed that the bacterial concentrations increased tremendously 3 days after infection, and the spleen was the major site for K. pneumoniae replication. The determination of the changes in tissue bacterial load provided useful data for elucidating the pathogenesis of K. pneumoniae in bullfrogs, while the mucus assay could be an effective tool for the early surveillance and prevention of K. pneumoniae in bullfrogs.

Detection of Old and New World Relapsing Fever Borreliae in Ornithodoros Ticks Collected from Warthog Burrows in Zambia.

Relapsing fever (RF) is an arthropod-borne disease caused by Borrelia spirochete, which is one of the major public health concerns in endemic regions including Africa. However, information on Borrelia spirochetes is limited in Zambia. Here, we investigate the Borrelia spirochetes harbored by Ornithodoros ticks in Zambian National Parks. We analyzed 182 DNA samples pooled from 886 Ornithodoros ticks. Of these, 43 tested positive, and their sequence revealed that the ticks harbored both Old and New World RF borreliae. This research presents the first evidence of Old-World RF borreliae in Zambia. The New World RF borreliae detected herein differed from the Candidatus Borrelia fainii previously reported in Zambia and were closely related to the pathogenic Borrelia sp. VS4 identified in Tanzania. Additionally, Borrelia theileri was recently reported in Zambia. Hence, at least four different Borrelia species occur in Zambia, and the organisms causing relapsing fever there might be more complex than previously thought. We empirically confirmed that real-time PCR with TaqMan minor groove binder probes accurately and simultaneously detected both Old and New World RF. In this manner, they could facilitate quantitative analyses of both types of RF borreliae. Subsequent investigations should endeavor to isolate the aforementioned Borrelia spp. and perform serosurveys on patients with RF.

Comparison of droplet digital PCR and real-time quantitative PCR for quantitative detection of the parasitic ciliate Ichthyophthirius multifiliis in the water environment.

Ichthyophthiriasis, caused by the parasitic ciliate Ichthyophthirius multifiliis (Ich), is considered one of the most harmful diseases affecting freshwater fish globally. It can cause mass mortalities of fish in intensive farming systems. In such systems, it is thus necessary to detect and quantify the number of Ich in the water so that control measures can be implemented before Ichthyophthiriasis breaks out. In recent years, molecular diagnostic methods have become increasingly important in aquaculture. Real-time quantitative polymerase chain reaction (qPCR) and droplet digital polymerase chain reaction (ddPCR) have become robust assays for detecting pathogens. In this study, a set of specific primers and a TaqMan-minor groove binder probe targeting the small-subunit rDNA (SSU rDNA) of Ich were developed. They were used in qPCR and ddPCR assays to compare the performance of these two different methods in quantitatively detecting Ich. After optimizing the reaction conditions, both qPCR and ddPCR assays were found to have high linearity and quantitative correlations for standard plasmid DNA. When used for the detection of Ich eDNA in water samples, the qPCR assay had a wider detection range, making it a suitable method to screen for the prevalence of Ichthyophthiriasis. However, the ddPCR approach had higher sensitivity, which would help provide advance notice of the disease in complex water environmental samples.

Detection of Pork DNA on Commercially Processed Meat Products Using Taqman qPCR for Label Verification

Research has been conducted to detect pork DNA in various commercially processed meat products. This study aims to analyze the content of pork DNA in different commercially processed meat products sold online in Jakarta using a Taqman minor groove binder probe combined with a brief profile of a qPCR machine to verify labels. The sample used in this study consisted of 5 types of processed meat products: canned corned, jerky, shredded, meatballs, and smoked meat. A total of 30 samples had no pork labels, and the five others had pork labels. All products were extracted, the concentration and the purity of DNA were measured, and fragmentation DNA and amplification were analyzed using Taqman qPCR. The results showed that the DNA concentration obtained was 14.02 – 176.04 ng/μL, DNA purity was obtained from 1.70 to 1.92, and all genomic DNA was fragmented. There was no amplification in 30 samples without pork labels. Five samples of pork products underwent PCR amplification with a Ct value of 22.96, 20.72, 26.78, 21.96, and 23.16 for canned corned, jerky, shredded pork meatballs, and smoked pork, respectively. The conclusion is that commercially processed meat products sold online in Jakarta comply with labels regulation by Indonesia Food and Drug Authority and consistently maintain the halalness of their products. The novelties in this study tested various commercially processed meat products sold online in Jakarta.

Development of a Triplex Real-Time PCR Assay to Detect Echinococcus Species in Canid Fecal Samples

Echinococcosis is a zoonotic disease with great significance to public health, and appropriate detection and control strategies should be adopted to mitigate its impact. Most cases of echinococcosis are believed to be transmitted by the consumption of food and/or water contaminated with canid stool containing Echinococcus spp. eggs. Studies assessing Echinococcus multilocularis, Echinococcus granulosus sensu stricto, and Echinococcus shiquicus coinfection from contaminated water-derived, soil-derived, and food-borne samples are scarce, which may be due to the lack of optimized laboratory detection methods. The present study aimed to develop and evaluate a novel triplex TaqMan-minor groove binder probe for real-time polymerase chain reaction (rtPCR) to simultaneously detect the 3 Echinococcus spp. mentioned above from canid fecal samples in the Qinghai-Tibetan Plateau area (QTPA). The efficiency and linearity of each signal channel in the triplex rtPCR assay were within acceptable limits for the range of concentrations tested. Furthermore, the method was shown to have good repeatability (standard deviation ≤0.32 cycle threshold), and the limit of detection was estimated to be 10 copies plasmid/μl reaction. In summary, the evaluation of the present method shows that the newly developed triplex rtPCR assay is a highly specific, precise, consistent, and stable method that could be used in epidemiological investigations of echinococcosis.

Rapid detection of H146-like goose calicivirus using real-time RT-PCR with a Taqman minor groove binder probe

H146-like goose-origin calicivirus (H146-like GCV) is a novel Caliciviridae family member in the Sanovirus genus that was associated with gosling growth retardation syndrome growth retardation syndrome complicated by visceral urate deposition. However, there is no accurate and high throughput real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) available for the rapid and highly sensitive identification of H146-like GCV. In this study, a pair of specific primers and a TaqMan minor groove binder (MGB) probe were designed based on a conserved region in the nonstructural (NS) gene sequence. The TaqMan-MGB probe-based one-step qRT-PCR assay was capable of detecting quite low number of targeting nucleic acid as low as 5.07 copies/μL and had excellent intra-assay and inter-assay repeatability with the coefficient of variation (CV) value from 0.558% to 1.293%. The assay was highly specific for H146-like GCV, without cross-reactions with other non-targeted goose-origin viruses, and 62 suspicious tissue samples infected with H146-like GCV from different regions of Fujian Province were used in this study to verify the feasibility and effectiveness of this assay in clinical diagnosis. The results indicated that our assay for the diagnosis and quantification of H146-like GCV was highly sensitive and specific, and should provide a reliable real-time tool for epidemiological and pathogenetic study of H146-like GCV infection, enabling researchers to better understand the epidemiology and clinical presentation of this disease.

Development and evaluation of a TaqMan MGB RT-PCR assay for detection of H5 and N8 subtype influenza virus

BackgroundHighly pathogenic influenza A (H5N8) viruses have caused several worldwide outbreaks in birds and are of potential risk to humans. Thus, a specific, rapid and sensitive method for detection is urgently needed.MethodsIn the present study, TaqMan minor groove binder probes and multiplex real-time RT-PCR primers were designed to target the H5 hemagglutinin and N8 neuraminidase genes. A total of 38 strains of avian influenza viruses and other viruses were selected to test the performance of the assay.ResultsThe results showed that only H5 and N8 avian influenza viruses yielded a positive signal, while all other subtypes avian influenza viruses and other viruses were negative. High specificity, repeatability, and sensitivity were achieved, with a detection limit of 10 copies per reaction.ConclusionsThe developed assay could be a powerful tool for rapid detection of H5N8 influenza viruses in the future.

Molecular genotyping and epidemiology of equine piroplasmids in South Africa

Recently reported substantial genetic diversity within Theileria equi 18S rRNA gene sequences has led to the identification of five genotypes A, B, C, D, and E, complicating molecular and serological diagnosis. In addition, T. haneyi has lately been reported as a species closely related to the T. equi 18S rRNA genotype C (Knowles et al., 2018). Theileria spp. of this group have a monophyletic origin and are therefore referred to as Equus group to distinguish them from the remaining Theileria lineages (Jalovecka et al., 2019). In this study, we report on the development of genotype-specific quantitative real-time PCR assays capable of detecting and distinguishing between each parasite genotype. Alignment of complete 18S rRNA sequences available on GenBank allowed for the design of a single primer pair and five TaqMan minor groove binder (MGB™) probes specific for each genotype (A–E). The assays, evaluated as qPCR simplex and two qPCR multiplex formats (Multiplex EP–ABC and Multiplex EP–DE), were shown to be both efficient and specific in the detection of T. equi genotypes. The developed qPCR assays were used to study (i) the intra-specific diversity of parasite genotypes within horse and zebra, (ii) the inter-specific differences in parasite genotype diversity in horses as compared to zebra, and (iii) the geographic distribution of T. equi 18S rRNA genotypes in South Africa. In addition, (iv) the presence of T. haneyi in South Africa was evaluated. An assessment of 342 equine field samples comprising 149 field horses, 55 racehorses, and 138 wild zebra confirmed the previously reported presence of T. equi 18S rRNA genotypes A, B, C, and D, and absence of genotype E in South African equids. Theileria equi genotypes A, B, C, and D, were detected in zebra, whereas only genotypes A, C and D, could be identified in field horses, and only genotypes A and C in racehorses. Genotypes B and D were the dominant genotypes identified in zebra in South Africa, while horses were predominantly infected with T. equi genotypes A and C. The greater diversity of T. equi genotypes in zebra suggests that it is an ancestral host for this piroplasmid lineage. Importantly, evidence is presented that each identified T. equi genotype segregates independently in each of the three studied equid populations reinforcing the notion that they represent individual separate entities corresponding to species. Preliminary investigations of the relationship between T. equi genotype C infections and Theileria haneyi, suggest that in addition to the five currently known T. equi genotypes, South African equids are also infected with T. haneyi.

Real-time reverse transcription polymerase chain reaction development for rapid detection of Tomato brown rugose fruit virus and comparison with other techniques.

BackgroundTomato brown rugose fruit virus (ToBRFV) is a highly infectious tobamovirus that causes severe disease in tomato (Solanum lycopersicum L.) crops. In Italy, the first ToBRFV outbreak occurred in 2018 in several provinces of the Sicily region. ToBRFV outbreak represents a serious threat for tomato crops in Italy and the Mediterranean Basin.MethodsMolecular and biological characterisation of the Sicilian ToBRFV ToB-SIC01/19 isolate was performed, and a sensitive and specific Real-time RT-PCR TaqMan minor groove binder probe method was developed to detect ToBRFV in infected plants and seeds. Moreover, four different sample preparation procedures (immunocapture, total RNA extraction, direct crude extract and leaf-disk crude extract) were evaluated.ResultsThe Sicilian isolate ToB-SIC01/19 (6,391 nt) showed a strong sequence identity with the isolates TBRFV-P12-3H and TBRFV-P12-3G from Germany, Tom1-Jo from Jordan and TBRFV-IL from Israel. The ToB-SIC01/19 isolate was successfully transmitted by mechanical inoculations in S. lycopersicum L. and Capsicum annuum L., but no transmission occurred in S. melongena L. The developed real-time RT-PCR, based on the use of a primer set designed on conserved sequences in the open reading frames3, enabled a reliable quantitative detection. This method allowed clear discrimination of ToBRFV from other viruses belonging to the genus Tobamovirus, minimising false-negative results. Using immunocapture and total RNA extraction procedures, the real-time RT-PCR and end-point RT-PCR gave the same comparable results. Using direct crude extracts and leaf-disk crude extracts, the end-point RT-PCR was unable to provide a reliable result. This developed highly specific and sensitive real-time RT-PCR assay will be a particularly valuable tool for early ToBRFV diagnosis, optimising procedures in terms of costs and time.

Correlations of IL-6 and CRP gene polymorphisms with pulmonary heart disease.

To explore the correlations of interleukin-6 (IL-6) and C-reactive protein (CRP) gene polymorphisms with pulmonary heart disease (PHD). A total of 98 patients with PHD and 102 healthy persons receiving physical examinations were enrolled. Their general clinical information was collected, and the levels of IL-6 and CRP in the plasma were determined. The pulmonary functions and blood gas were detected, and the TaqMan-minor groove binder (MGB) probe was used to detect the polymorphisms of IL-6 rs1800796 and CRP rs1800796. Observation group had higher levels of IL-6 and CRP than control group (p<0.05). The forced expiratory volume in 1 second (FEV1) (%), FEV1/forced vital capacity (FVC) ratio (%), and arterial partial pressure of oxygen (PaO2) in observation group were lower than those in control group (p<0.05), but the arterial partial pressure of carbon dioxide (PaCO2) was higher than that in control group (p<0.05). There were differences in the distribution frequencies of the genotypes and alleles of IL-6 rs1800796 and CRP rs1800796 between the two groups (p<0.05). IL-6 and CRP are correlated with the onset of PHD, and there are also correlations between the polymorphisms of IL-6 rs1800796 and CRP rs2794521 and the disease.

Detection and quantification of Aeromonas schubertii in Channa maculata by TaqMan MGB probe fluorescence real-time quantitative PCR.

Aeromonas schubertii is a major epidemiological agent that threatens cultured snakeheads (Channidae) and has caused great economic losses in fish-farming industries in China in recent years. In present study, a specific TaqMan minor groove binder (MGB) probe fluorescence real-time quantitative PCR (qPCR) assay was developed to rapidly detect and quantify A.schubertii. A pair of qPCR primers and a TaqMan MGB probe were selected from the rpoD gene, which were shown to be specific for A.schubertii. A high correlation coefficient (R2 =0.9998) in a standard curve with a 103% efficiency was obtained. Moreover, the qPCR method's detection limit was as low as 18copies/μl, which was 100 times more sensitive than that of conventional PCR. The detection results for the A.schubertii in pond water and fish tissue were consistent with those of the viable counts. Bacterial load changes detected by qPCR in different tissues of snakeheads infected with A.schubertii showed that the gills and intestines may be the entry for A.schubertii, and the spleen and kidney are major sites for A.schubertii replication. The established method in present study should be a useful tool for the early surveillance and quantitation of A.schubertii.

Development of a TaqMan MGB RT-PCR assay for the detection of type A and subtype H10 avian influenza viruses.

H10 subtype avian influenza viruses have caused several epidemics in poultry and mammals, and specific, rapid and sensitive methods for detection are urgently needed. Herein, TaqMan minor groove binder (MGB) probes and multiplex real-time RT-PCR primers were designed based on gene regions encoding conserved domains of the nucleoprotein and H10 hemagglutinin. The developed multiplex real-time RT-PCR assay displayed high specificity, repeatability, and a detection limit of 10 copies per reaction. This diagnostic method could prove valuable for the rapid detection of H10 subtype AIVs in China.

Detection of KIT Genotype in Pigs by TaqMan MGB Real-Time Quantitative Polymerase Chain Reaction.

The dominant white phenotype in domestic pigs is caused by two mutations in the KIT gene: a 450 kb duplication containing the entire KIT gene together with flanking sequences and one splice mutation with a G:A substitution in intron 17. The purpose of this study was to establish a simple, rapid method to determine KIT genotype in pigs. First, to detect KIT copy number variation (CNV), primers for exon 2 of the KIT gene, along with a TaqMan minor groove binder (MGB) probe, were designed. The single-copy gene, estrogen receptor (ESR), was used as an internal control. A real-time fluorescence-based quantitative PCR (FQ-PCR) protocol was developed to accurately detect KIT CNVs. Second, to detect the splice mutation ratio of the G:A substitution in intron 17, a 175 bp region, including the target mutation, was amplified from genomic DNA. Based on the sequence of the resulting amplified fragment, an MGB probe set was designed to detect the ratio of splice mutation to normal using FQ-PCR. A series of parallel amplification curves with the same internal distances were obtained using gradually diluted DNA as templates. The CT values among dilutions were significantly different (p < 0.001) and the coefficients of variation from each dilution were low (from 0.13% to 0.26%). The amplification efficiencies for KIT and ESR were approximately equal, indicating ESR was an appropriate control gene. Furthermore, use of the MGB probe set resulted in detection of the target mutation at a high resolution and stability; standard curves illustrated that the amplification efficiencies of KIT1 (G) and KIT2 (A) were approximately equal (98.8% and 97.2%). In conclusion, a simple, rapid method, with high specificity and stability, for the detection of the KIT genotype in pigs was established using TaqMan MGB probe real-time quantitative PCR.

Survey of Potential Reservoir Species for the Oyster Parasite Multinucleate Sphere X (Haplosporidium nelsoni) in and Around Oyster Farms in the Damariscotta River Estuary, Maine

ABSTRACT In the summer of 2010, the Damariscotta River Estuary, ME, experienced the first large multinucleate sphere X disease (Haplosporidium nelsoni) outbreak in commercial stocks of the eastern oyster (Crassostrea virginica). In 2012, biofouling organisms were sampled in and around the commercial oyster operations with the intent of looking for the presence of the parasite to see which, if any, biofouling species may be acting as a reservoir host and harboring the parasite. Organisms collected included tunicates, gastropods, polychaetes, and arthropods, as well as plankton samples from the surrounding water column. A quantitative polymerase chain reaction assay for H. nelsoni using a TaqMan minor groove binder probe, which was originally developed for detection of the parasiteDNAin oysters, was applied to biofouling species. The protozoan parasite H. nelsoniDNA was present in up to 70% of tunicate samples and about 30% of plankton samples. The highest parasite DNA copies (averaging 4 × 104 copies) were...

Role of UMOD Promoter Polymorphism in the Etiology of Preeclampsia

To evaluate the association between preeclampsia (PE) and the single nucleotide polymorphism (SNP) rs13333226, located in the promoter region of the UMOD gene. A total of 1248 pregnant Han Chinese women (716 controls and 532 patients with PE) were included in this study. Genotyping of the rs13333226 polymorphism was performed by real-time PCR using a TaqMan-minor groove binder (MGB) probe assay. No significant differences were detected in the allele (p = 0.62, OR = 1.08, 95% CI = 0.81-1.44) and genotype frequencies of rs13333226 (padditive = 0.38, pdominant = 0.45, precessive = 0.31) between cases and controls. When patients were divided into subgroups, no association was found with mild preeclampsia (M PE), severe preeclampsia (S PE), early onset PE, or late-onset PE. Furthermore, no significant differences were detected in the genotype and allele frequencies of rs1333226 between patients with M PE and S PE (p > 0.05) or between patients with late and early onset of the disease (p > 0.05). UMOD rs13333226 does not appear to be associated with PE in Han Chinese women.

Development of a real-time quantitative PCR assay using a TaqMan minor groove binder probe for the detection of α-lactalbumin in food

Here, we report the development of a real-time PCR assay using a TaqMan minor groove binder (MGB, Genecore, NCBI: AF249896.1, 806-820) probe and primer sets designed to recognize the α-lactalbumin gene from the cow (Bos taurus). We evaluated the efficacy of this assay for detecting and quantifying cow α-lactalbumin in commercial foods. Our results demonstrated that the developed method was highly sensitive and showed high specificity for cow milk, with consistent detection of 0.05 ng of bovine DNA. We tested 42 commercial food samples with or without cow milk listed as an ingredient by using the developed assay. Among the 42 samples, 26 products that listed milk as an ingredient and 3 products might contain milk showed positive signals, whereas the other 9 products that did not contain milk and 4 products that might contain milk tested negative. Therefore, this method could be widely used for the detection of cow milk allergens in food.

A simplified duplex real-time PCR incorporating TaqMan minor groove binder (MGB) probes and an exogenous internal positive control for the simultaneous detection of Mycoplasma gallisepticum and Mycoplasma synoviae cultures

Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) are the most pathogenic and economically important mycoplasma pathogens that infect chickens. The development of rapid and innovative molecular diagnostic techniques is of pivotal importance for their effective control. The aim of the present study was to develop a novel duplex TaqMan real-time PCR assay for the simultaneous detection of MG and MS. This duplex real-time PCR assay incorporates TaqMan (FAM/NED) labelled minor groove binder (MGB) probes that target the cytadhesin encoding surface protein (mgc2) gene and the haemagglutinin surface protein (vlhA) gene of MG and MS, respectively. The assay also contained a TaqMan exogenous internal positive control (Exo IPC), to avoid false negative results that might happen due to failure in DNA extraction/PCR inhibition. The TaqMan MGB probe-based duplex RT-PCR incorporating Exo IPC was then applied to DNA from culture isolates for the simultaneous detection of MG (mgc2 gene) and MS (vlhA gene). For duplex RT-PCR the sensitivity recorded was 10-3 CFU/ml and 10-2 CFU/ml for MG and MS template DNA, respectively. The specificity of the real-time PCR assay was 100% for MG- and MS-specific probes in detecting both single as well as double infections. In conclusion, the use of TaqMan MGB probes for the detection of mgc2 and vlhA genes confers extra specificity and the incorporation of Exo IPC simplifies the assay, allowing the detection of double infections with low-copy target DNAs.