SYBR Green Real-time PCR Assay Research Articles

Introducing and validation of SYBR Green Real-Time PCR method to determinate sex ratio in bovine semen

Flow cytometry is a widely used application for validating the accuracy of sperm sexing. However, this method is relatively expensive and requires considerable technical support. An alternative method employing simpler technology at low cost could be suitable for the evaluation of bovine semen in laboratories with low budgets. We used a SYBR Green Real-Time PCR assay to determinate sex ratio in bovine semen. The PLP and SRY genes were amplified to isolate the specific fragments of X- and Y-chromosome sequences, respectively. Two certified standard curves were obtained using two plasmids containing PLP and SRY amplicons. Our results show no significant difference in semen sex ratio in unsorted semen (54.7±0.52% X and 47.6±0.60% Y). However, significant difference was observed in X/Y-sorted semen (93.3±0.08% X and 91.4±0.06% Y-sperm), as compared to the expected ratio in unsorted semen or the post-sorting reanalysis data. The evolution of X-chromosome bearing sperm content in unsorted samples showed an average of 52.6 for ejaculates and 51.8 for the commercial semen. In order to confirm our results, the accuracy, repeatability and reproducibility of the method were tested resulting in 98.2% accuracy, repeatability of CV=5.59% and reproducibility of CV=5.40%. Thus, this method is demonstrated to be a reliable and inexpensive way to test sexual chromosome content in semen samples.

First detection of Waddlia chondrophila in Africa using SYBR Green real-time PCR on veterinary samples

Waddlia chondrophila is a strict intracellular microorganism belonging to the order Chlamydiales that has been isolated twice from aborted bovine fetuses, once in USA and once in Germany. This bacterium is now considered as an abortigenic agent in cattle. However, no information is available regarding the presence of this bacterium in Africa. Given the low sensitivity of cell culture to recover such an obligate intracellular bacterium, molecular-based diagnostic approaches are warranted. This report describes the development of a quantitative SYBR Green real-time PCR assay targeting the recA gene of W. chondrophila. Analytical sensitivity was 10 copies of control plasmid DNA per reaction. No cross-amplification was observed when testing pathogens that can cause abortion in cattle. The PCR exhibited a good intra-run and inter-run reproducibility. This real-time PCR was then applied to 150 vaginal swabs taken from Tunisian cows that have aborted. Twelve samples revealed to be Waddlia positive, suggesting a possible role of this bacterium in this setting. This new real-time PCR assay represents a diagnostic tool that may be used to further study the prevalence of Waddlia infection.

A SYBR Green real-time PCR assay to detect and quantify pork meat in processed poultry meat products

Species identification in meat products has grown in interest in recent years since these foodstuffs are susceptible targets for fraudulent labelling. In this work, a real-time PCR approach based on SYBR Green dye was proposed for the quantitative detection of pork meat in processed meat products. For the development of the method, binary meat mixtures containing known amounts of pork meat in poultry meat were used to obtain a normalised calibration model from 0.1 to 25% with high linear correlation and PCR efficiency. The method revealed high specificity by melting curve analysis, being successfully validated through its application to blind meat mixtures, which confirmed its adequacy for pork meat determination. The fully applicability of the method was further demonstrated in commercial meat products, allowing verification of labelling compliance and identification of meat species in processed foods.

Hyperlipidemia offers protection against Leishmania donovani infection: role of membrane cholesterol

Leishmania donovani (LD), the causative agent of visceral leishmaniasis (VL), extracts membrane cholesterol from macrophages and disrupts lipid rafts, leading to their inability to stimulate T cells. Restoration of membrane cholesterol by liposomal delivery corrects the above defects and offers protection in infected hamsters. To reinforce further the protective role of cholesterol in VL, mice were either provided a high-cholesterol (atherogenic) diet or underwent statin treatment. Subsequent LD infection showed that an atherogenic diet is associated with protection, whereas hypocholesterolemia due to statin treatment confers susceptibility to the infection. This observation was validated in apolipoprotein E knockout mice (AE) mice that displayed intrinsic hypercholesterolemia with hepatic granuloma, production of host-protective cytokines, and expansion of antileishmanial CD8(+)IFN- γ (+) and CD8(+)IFN- γ (+)TNF- α (+) T cells in contrast to the wild-type C57BL/6 (BL/6) mice when infected with LD. Normal macrophages from AE mice (N-AE-MΦ) showed 3-fold higher membrane cholesterol coupled with increased fluorescence anisotropy (FA) compared with wild-type macrophage (N-BL/6-MΦ). Characterization of in vitro LD-infected AE macrophage (LD-AE-MΦ) revealed intact raft architecture and ability to stimulate T cells, which were compromised in LD-BL/6-MΦ. This study clearly indicates that hypercholesterolemia, induced intrinsically or extrinsically, can control the pathogenesis of VL by modulating immune repertoire in favor of the host.

Quantitative in planta PCR assay for specific detection of Xanthomonas oryzae pv. oryzicola using putative membrane protein based primer set

Molecular-based techniques are becoming desirable as tools for identification and detection of plant diseases. Amongst the Xanthomonas oryzae pathovars, there is a need to differentiate X. oryzae pv. oryzicola from X. oryzae pv. oryzae, as misidentification could lead to improper treatment of rice crops. Therefore, we developed a novel SYBR green real-time PCR assay to target a putative membrane protein gene of X. oryzae pv. oryzicola BLS256. The specificity of the primer set was tested using purified DNA from eleven X. oryzae pv. oryzicola strains, three X. oryzae pv. oryzae strains, and twenty-six other reference pathogenic bacteria. The primer set amplified a single band of expected size from genomic DNA obtained from X. oryzae pv. oryzicola strains, but not from X. oryzae pv. oryzae or from other bacterial strains. The assay was also able to detect at least two genome equivalents of cloned amplified target DNA using purified DNA, or 0.1 × 10−5 OD600 unit of cells per reaction, respectively. This assay represents a novel approach to bacterial leaf streak diagnosis that will advance the field of rice disease research.

Dual Priming Oligonucleotides for Broad-Range Amplification of the Bacterial 16S rRNA Gene Directly from Human Clinical Specimens

Broad-range amplification and sequencing of the bacterial 16S rRNA gene directly from clinical specimens are offered as a diagnostic service in many laboratories. One major pitfall is primer cross-reactivity with human DNA which will result in mixed chromatograms. Mixed chromatograms will complicate subsequent sequence analysis and impede identification. In SYBR green real-time PCR assays, it can also affect crossing threshold values and consequently the status of a specimen as positive or negative. We evaluated two conventional primer pairs in common use and a new primer pair based on the dual priming oligonucleotide (DPO) principle. Cross-reactivity was observed when both conventional primer pairs were used, resulting in interpretation difficulties. No cross-reactivity was observed using the DPOs even in specimens with a high ratio of human to bacterial DNA. In addition to reducing cross-reactivity, the DPO principle also offers a high degree of flexibility in the design of primers and should be considered for any PCR assay intended for detection and identification of pathogens directly from human clinical specimens.

Quantification of Manila ClamRuditapes philippinarum(Adams & Reeve, 1850) Larvae Based on SYBR Green Real-Time Polymerase Chain Reaction

Biological and ecological research is seriously handicapped because of difficulties experienced in the reliable detection and quantification of bivalve larvae. This is a critical issue in the case of the Manila clam Ruditapes philippinarum (Adams & Reeve, 1850), a largely invasive and commercially relevant species, with important wild, cultured, and naturalized populations throughout the world. A SYBR Green real-time PCR assay, containing TPHI16S1F and TPHI16S2R primers (specific to R. philippinarum female mtDNA), was designed and tested to provide a rapid and high-throughput PCR-based method for larval quantification. Accurate estimations of larval numbers in spiked plankton samples point to the usefulness of this system. It can be used through a wide range of evaluated variable experimental conditions, such as the presence of closely related bivalve species, stationary-dependent plankton abundance, sampling volumes, and larval size.

A Real-Time PCR Assay for Bat SARS-Like Coronavirus Detection and Its Application to Italian Greater Horseshoe Bat Faecal Sample Surveys

Bats are source of coronaviruses closely related to the severe acute respiratory syndrome (SARS) virus. Numerous studies have been carried out to identify new bat viruses related to SARS-coronavirus (bat-SARS-like CoVs) using a reverse-transcribed-polymerase chain reaction assay. However, a qualitative PCR could underestimate the prevalence of infection, affecting the epidemiological evaluation of bats in viral ecology. In this work an SYBR Green-real time PCR assay was developed for diagnosing infection with SARS-related coronaviruses from bat guano and was applied as screening tool in a survey carried out on 45 greater horseshoe bats (Rhinolophus ferrumequinum) sampled in Italy in 2009. The assay showed high sensitivity and reproducibility. Its application on bats screening resulted in a prevalence of 42%. This method could be suitable as screening tool in epidemiological surveys about the presence of bat-SARS-like CoVs, consequently to obtain a more realistic scenario of the viral prevalence in the population.

Quantitative and Qualitative Analyses of the SNRPN Gene Using Real-Time PCR with Melting Curve Analysis

Prader-Willi syndrome and Angelman syndrome are distinct neurodevelopmental disorders that are associated with the deletion of the chromosomal 15q11-13 region or uniparental disomy of chromosome 15. In this article, we applied SYBR Green I-based real-time PCR and melting curve analysis assay for rapid genotyping of the small nuclear ribonucleoprotein polypeptide N (SNRPN) gene methylation status and for detecting aberrations in copy number in a single tube. A single pair of primers was designed to create a 357 bp fragment containing the cytosine phosphodiester guanine islands in the SNRPN promoter and to amplify both unmethylated and methylated sequences. Genotypes were identified based on the TC value for copy number changes and the characteristic melting temperature of methylated cytosine phosphodiester guanine. Genotyping of SNRPN was performed on blood samples of 20 individuals with Prader-Willi syndrome, 3 individuals with Angelman syndrome, and 20 unaffected individuals. The promoter methylation status and the copy number changes were successfully determined and compared with standard methylation-specific PCR, and were validated by multiplex ligation-dependent probe amplification. This single-tube, SYBR Green I, real-time PCR with melting curve assay is rapid, reliable, sensitive, and easy to perform. It is suitable for high-throughput analysis as an alternative technique for quantitative and qualitative analysis of target genes.

Detection and species identification of microsporidial infections using SYBR Green real-time PCR

Diagnosis of microsporidial infections is routinely performed by light microscopy, with unequivocal non-molecular species identification achievable only through electron microscopy. This study describes a single SYBR Green real-time PCR assay for the simultaneous detection and species identification of such infections. This assay was highly sensitive, routinely detecting infections containing 400 parasites (g stool sample)(-1), whilst species identification was achieved by differential melt curves on a Corbett Life Science Rotor-Gene 3000. A modification of the QIAamp DNA tissue extraction protocol allowed the semi-automated extraction of DNA from stools for the routine diagnosis of microsporidial infection by real-time PCR. Of 168 stool samples routinely analysed for microsporidian spores, only five were positive by microscopy. By comparison, 17 were positive for microsporidial DNA by real-time analysis, comprising 14 Enterocytozoon bieneusi, one Encephalitozoon cuniculi and two separate Pleistophora species infections.

Development of a real-time SYBRGreen PCR assay for rapid detection of acquired AmpC in Enterobacteriaceae

IntroductionAcquired AmpC enzymes, classified as miscellaneous extended-spectrum β-lactamase (ESBLM) enzymes according to a recently proposed β-lactamase classification, are increasing according to several publications. Simple and rapid methods for detection of ESBLM are needed for appropriate infection control. A gel-based multiplex PCR method for acquired blaAmpC detection and subtype classification has been available for several years. Here, we describe a modification of the protocol to suit real-time PCR platforms and to include novel genotypes. Material and methodsClinical isolates with clavulanic acid non-reversible non-susceptibility to extended-spectrum cephalosporins were subjected to combination disk testing with cefoxitin +/− cloxacillin at Malmö University Hospital. Phenotypical AmpC production was defined as cloxacillin reversible cefoxitin resistance. In this study 51 phenotypical AmpC-producing isolates, were subjected to the acquired blaAmpC real-time PCR assay. The acquired blaAmpC positive isolates were further characterized by DNA sequencing of the acquired AmpC encoding gene, Pulsed-Field Gel Electrophoresis (PFGE) and PCR-based replicon typing. Results and discussionThe real-time PCR assay was able to detect and sub-classify all acquired blaAmpC genes described to date. The assay can be performed in less than 3h, including pre-PCR preparations. Analysis of the isolate collection resulted in 18 of 51 phenotypical AmpC-producing isolates being positive in the acquired blaAmpC real-time multiplex PCR assay; 17 of subtype CIT and one DHA. Sequence analysis identified 16 isolates as blaCMY-2, one as blaCMY-16 and one as blaDHA-1. Detected plasmid replicon types were I1 and B/O. Two of the E. coli isolates were identical according to PFGE and the others were unrelated.

Quantitative diagnosis and analysis of mutations affecting drug resistance to rifampicin and isoniazid of clinical Mycobacterium tuberculosis isolates in Taiwan

A SYBR Green I-based real-time quantitative PCR assay was utilized using senX3-regX3 and IS6110 as targets to identify Mycobacterium tuberculosis in 135 clinical samples from southern Taiwan. The bacterial loads of true positive samples estimated in this assay ranged from 33 to 7.7 × 105 CFU/ml, whereas false negative samples ranged from 10 to 25 CFU/ml. The specificity and sensitivity were 100% and 88.5%, respectively. Moreover, a multiplex allele-specific PCR was applied to detect mutation at three major hot spots (affecting codons 516, 526 and 531) of the rpoB gene affecting resistance to rifampicin, as well as codon 315 of the katG gene (resistance to isoniazid). The mutation rates of rpoB and katG were 69.6% (32/46) and 45.7% (21/46), respectively. The dual-gene mutation (rpoB and katG) rate was 41.3% (19/46). Among the hot spots on the rpoB gene, the mutation rate for rpoB 531(17/46, 37%) was higher than that of rpoB 516 (10/46, 21.7%) and rpoB 526 (8/46, 17.4%). Furthermore, for the four codons assayed (rpoB 516, 526, 531, and katG), 11 mutations were identified as single mutation (23.9%), 18 as double mutations (39.1%), 3 as triple mutations, and none as quadruple mutations. Our results reveal the prevalence of tuberculosis drug-resistant mutations occurring on rpoB and katG genes in southern Taiwan.

Detection of BPV-1 and -2 and Quantification of BPV-1 by Real-Time PCR in Cutaneous Warts in Cattle and Buffaloes

Bovine cutaneous warts (CWs) were investigated in Northern India. Of 49 cases, 44 were recorded in cattle and 5 in buffaloes. These animals had mild to moderate grade infections. Grossly, cases of CWs appeared to be of exophytic type, however, different types of growth patterns were observed. A total of 26 biopsies (cattle 21 and buffaloes 5) from CWs-affected animals studied histopathologically were diagnosed as exophytic and cauliflower-like fibropapilloma 13, exophytic and dome-shaped fibropapilloma 5, occult and/or fibroblastic type papilloma 3, cauliflower-like papilloma 3, endophytic fibropapilloma 1 and fibroma 1. On PCR analysis, 11 CWs and 2 normal skin samples showed BPV-1, -2 mixed infections. A rapid, sensitive and reliable real-time SYBR Green PCR test to detect BPV-1, BPV-2 and to quantify BPV-1 was developed. Results of amplification and dissociation plot of real-time PCR revealed that six samples were BPV-1 positive, eight were BPV-2 positive and six were positive for both BPV-1 and -2. CWs samples from different dairy farms testing positive for BPV-1 by PCR assay were also positive using Quantitative real-time SYBR Green PCR assay. For the first time, mixed infection of BPV-1 and -2 was detected in India and BPV-1 load was quantified by real-time SYBR Green PCR assay.

Sjögren’s Syndrome-Like Ocular Surface Disease in Thrombospondin-1 Deficient Mice

Thrombospondin-1 (TSP-1) is a major activator of latent transforming growth factor-beta in vitro as well as in vivo. Mice deficient in TSP-1, despite appearing normal at birth, develop a chronic form of ocular surface disease that is marked by increased apoptosis and deterioration in the lacrimal gland, associated dysfunction, and development of inflammatory infiltrates that result in abnormal tears. The increase in CD4(+) T cells in the inflammatory infiltrates of the lacrimal gland, and the presence of anti-Sjögren's syndrome antigen A and anti-Sjögren's syndrome antigen B antibodies in the serum resemble autoimmune Sjögren's syndrome. These mice develop an ocular surface disorder dry eye that includes disruption of the corneal epithelial layer, corneal edema, and a significant decline in conjuctival goblet cells. Externally, several mice develop dry crusty eyes that eventually close. The inflammatory CD4(+) T cells detected in the lacrimal gland, as well as those in the periphery of older TSP-1 null mice, secrete interleukin-17A, a cytokine associated with chronic inflammatory diseases. Antigen-presenting cells, derived from TSP-1 null, but not from wild-type mice, activate T cells to promote the Th17 response. Together, these results indicate that TSP-1 deficiency results in a spontaneous form of chronic dry eye and aberrant histopathology associated with Sjögren's syndrome.

Development of real-time PCR systems based on SYBR Green for the specific detection and quantification of Klebsiella pneumoniae in infant formula

Klebsiella pneumoniae is an emerging opportunistic pathogen that causes severe morbidity and mortality among the newborn and immunocompromised persons. Infant formula has been associated with the K. pneumoniae-related outbreaks in a newborn intensive care unit. In this study, a SYBR Green real-time PCR assay was developed for the rapid identification of K. pneumoniae from infant formulas. 142-bp phoE gene was selected for specificity, and two K. pneumoniae strains and 15 non- K. pneumoniae strains were evaluated. All K. pneumoniae strains tested were phoE-positive to the real-time PCR detection and no cross-reactivity was observed with non- K. pneumoniae strains. Sensitivity of the detections is 1 CFU/25 g infant formulas. And the assays can be accomplished within two business days including pre-enrichment. The results indicate that the method is efficient and can be employed to detect specifically K. pneumoniae from infant formulas with considerable sensitivity. In this study, we also constructed reference standards for quantification of K. pneumoniae by real-time PCR. The values of the cycle threshold (Ct) were linearly correlated with the concentration of the reference standards, indicating that they can be used as quantitative detection of K. pneumoniae. This result is of special significance for applications of predicting the load of K. pneumoniae.

Molecular analysis of HPV 16 E6I/E6II spliced mRNAs and correlation with the viral physical state and the grade of the cervical lesion

The presence of HPV 16 E6*I/E6*II spliced transcripts, in cervical lesions of different grade, was analyzed to characterize the transcription pattern. The presence and amount of spliced transcripts were correlated with DNA viral markers such as E2/E6 ratio and physical state. The detection of HPV 16 E6*I/E6*II mRNAs was set up by an SYBR Green real-time reverse transcriptase PCR assay with an optimal dynamic range and sensitivity. The assay was applied to the analysis of 71 specimens, positive to HPV 16 as a sole infection, from women with abnormal cervical smears, precisely 31 low-grade squamous intraepithelial lesions and 40 high-grade lesions. Samples negative to both transcripts were found only in low-grade cervical lesions. Three different transcription profiles were found in the low- and high-grade lesions analyzed: in low-grade lesions samples positive only to E6*II and in high-grade lesions samples positive only to E6*I were detected. In low- and high-grade lesions, samples positive to both E6*I and E6*II were found. In the samples positive for both transcripts, the E6*I/E6*II ratio was higher than that in the majority of high-grade lesions and lower than that in all the low-grade lesions. Analyzing the transcription pattern in relation to E2/E6 ratio and to the DNA physical state, the presence of high values of E6*I was associated mainly with low values of E2/E6 ratio and of mixed DNA forms. The detection of HPV 16 E6*I/E6*II mRNAs may serve to identify transcription patterns indicative of cervical disease progression and help physicians to decide clinical management.

Design and testing of real-time PCR primers for the quantification ofMethanoculleus,Methanosarcina,Methanothermobacter, and a group of uncultured methanogens

In this study, 16S rRNA gene primers were designed to complement the suite of already available PCR primers for the detection of different methanogens involved in biogas production through anaerobic digestion by SYBR Green real-time PCR. Primers designed for use in TaqMan real-time PCR for the organisms Methanosaeta, Methanosarcina, and Methanoculleus have been described previously; however, we found that (i) the Methanoculleus primers were not specific to members of the genus and that (ii) the Methanosarcina primers did not work specifically with SYBR Green real-time PCR. Thus, we designed new primers for these and other methanogens, and we optimized SYBR Green real-time PCR assays. Primers were tested by end-point and real-time PCR, and they were found to work specifically and sensitively. Application of these primers will allow the detection and quantification of Methanoculleus, Methanosarcina, Methanothermobacter, and a group of yet uncultured archaea from anaerobic habitats.

Optimizing the yield and utility of circulating cell-free DNA from plasma and serum

Background Cell-free DNA (CFDNA) in the plasma/serum of patients with cancer demonstrates tumour-associated genetic alterations, offering possibilities for diagnosis, prognostication and disease monitoring. There is wide variation in the reported levels of CFDNA, associated with different methods used to collect, process and analyze blood samples. We therefore evaluated different aspects of laboratory protocols for the processing and purification of CFDNA in clinical studies. Methods We evaluated and compared the QIAamp kit and a Triton/Heat/Phenol protocol (THP) for CFDNA purification. Total CFDNA was quantified by PicoGreen assay and SYBR-Green real-time PCR assay was used to amplify specific genes to estimate the efficiency of different protocols. Results The efficiency of DNA extraction was 18.6% using the standard QIAamp protocol and 38.7% using the THP method ( p < 0.0001, unpaired t-test). A modified QIAamp protocol that included a proteinase incubation stage and elution volumes up to 300 μl increased DNA yields, but was not as good as the THP method. Conclusions Blood samples should be kept at/or below room temperature (18 °C–22 °C) for no more than 2 h before plasma separation by double-spin. Because of its higher efficiency, low-cost and good-quality products, the THP protocol is preferred for extraction of CFDNA.

Development of a Real-Time Polymerase Chain Reaction Assay for The Simultaneous Detection of Mycoplasma Gallisepticum and Mycoplasma Synoviae Under Industry Conditions

In this research we developed a real-time SYBR green assay to detect both Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) in a single reaction. A total of 30,000 samples from broiler breeder flocks were screened using traditional serology (plate agglutination, enzyme-linked immunosorbent assay, hemagglutination inhibition) and polymerase chain reaction (PCR; traditional and real-time). It was determined that the real-time SYBR green PCR assay developed in this research was more rapid than all three methods tested and more sensitive and specific than culturing or serology. The SYBR green assay was optimized and could detect as few as 30 template copies of DNA per sample. In addition, the SYBR green assay was less expensive than traditional culturing and serology. MG and MS are infectious bacteria that can rapidly spread and infect commercial chicken flocks. These diseases can cause a significant loss to the poultry industry and especially to broiler breeders because infected flocks are destroyed under the National Poultry Improvement Plan MG and MS clean programs. The real-time SYBR green assay developed in this research has the potential to reduce the time it takes to reach a correct diagnosis and to arrest outbreaks of MG and MS.

Development and comparison of SYBR Green quantitative real-time PCR assays for detection and enumeration of sulfate-reducing bacteria in stored swine manure

To develop and evaluate primer sets targeted to the dissimilatory sulfite reductase gene (dsrA) for use in quantitative real-time PCR detection of sulfate-reducing bacteria (SRB) in stored swine manure. Degenerate primer sets were developed to detect SRB in stored swine manure. These were compared with a previously reported primer set, DSR1F+ and DSR-R, for their coverage and ability to detect SRB communities in stored swine manure. Sequenced clones were most similar to Desulfovibrio sp. and Desulfobulbus sp., and these SRB populations differed within different manure ecosystems. Sulfur content of swine diets was shown to affect the population of Desulfobulbus-like Group 1 SRB in manure. The newly developed assays were able to enumerate and discern different groups of SRB, and suggest a richly diverse and as yet undescribed population of SRB in swine manure. The PCR assays described here provide improved and efficient molecular tools for quantitative detection of SRB populations. This is the first study to show population shifts of SRB in swine manure, which are a result of either the effects of swine diets or the maturity of the manure ecosystem.