Results Of Real-time PCR Assays Research Articles

Sampling Adipose and Muscle Tissue following Post-Harvest Scalding Does Not Affect RNA Integrity or Real-Time PCR Results in Market Weight Yorkshire Pigs.

Improving production efficiency while enhancing pork quality is pivotal for strengthening sustainable pork production. Being able to study both gene expression and indices of pork quality from the same anatomical location of an individual animal would better enable research conducted to study relationships between animal growth and carcass merit. To facilitate gene expression studies, adipose and muscle tissue samples are often collected immediately following exsanguination to maximize RNA integrity, which is a primary determinant of the sensitivity of RNA-based assays, such as real-time PCR. However, collecting soft tissue samples requires cutting through the hide or skin. This leaves the underlying tissue exposed during scalding, poses possible food safety issues, and potentially confounds pork quality measures. To overcome these limitations, the effect of tissue sample timing post-harvest on RNA integrity, real-time PCR results, and pork quality measurements was investigated by sampling subcutaneous adipose tissue and longissimus thoracis et lumborum muscle immediately following either exsanguination, scalding, or chilling. Sampling time did not affect RNA quality, as determined by the RNA integrity number of RNA samples purified from either adipose (RIN; p > 0.54) or muscle tissue (p > 0.43). Likewise, the sampling time did not influence the results of real-time PCR analysis of gene expression when comparing RNA samples prepared from adipose or muscle tissue immediately following either exsanguination or scalding (p > 0.92). However, sampling tissue prior to scalding resulted in a greater visual color score (p < 0.001) and lesser L* (p < 0.001) and b* (p < 0.001) values without impacting the 24 h pH (p < 0.41). These results suggested that if both RNA-based assays and meat quality endpoints are to be performed at the same anatomical location on an animal, tissue sampling to facilitate RNA-based assays should occur at a time point immediately following scalding. These findings demonstrated that sampling of adipose and muscle tissue can be delayed until after scalding/dehairing without decreasing the RNA integrity or altering the results of real-time PCR assays, while doing so was associated with little impact on measures of pork quality.

Evaluation of an automated insulated isothermal polymerase chain reaction system for rapid and reliable, on-site detection of African swine fever virus.

To evaluate the utility of an automated insulated isothermal PCR (iiPCR) system for rapid and reliable on-site detection of African swine fever virus (ASFV) in swine biological samples. Lymph node, tissue homogenate, whole blood, serum, spleen, and tonsil samples collected from swine in North and South Vietnam. Analytic sensitivity of the iiPCR system was determined by serial dilution and analysis of 2 samples (swine tissue homogenate and blood) predetermined to be positive for ASFV. Analytic specificity was assessed by analysis of 2 samples predetermined to be negative for ASFV and positive or negative for other swine pathogens (classical swine fever virus, porcine reproductive and respiratory syndrome virus, foot-and-mouth disease virus, and porcine circovirus type 2). Diagnostic performance of the iiPCR system for detection of ASFV was determined by analysis of the various tissue sample types. For all tests, a real-time PCR assay was used as the reference method. The iiPCR system was able to detect ASFV in swine blood or tissue homogenate at dilutions up to 106, whereas the real-time PCR assay was able to detect dilutions of up to 105 or 106. The iiPCR system had high analytic specificity for detection of ASFV versus other swine pathogens. Between 97% and 100% agreement was found between results of the iiPCR system for the various tissue samples and results of real-time PCR assay. The evaluated iiPCR system was found to be a rapid, reliable, and sample-flexible method for ASFV detection and may be useful for disease surveillance and quarantine in national strategies for early ASF control.

In vitro propagation of infectious myonecrosis virus in C6/36 mosquito cell line.

Infectious myonecrosis (IMN) is an important shrimp viral disease caused by infectious myonecrosis virus (IMNV). Based on previous reports, an attempt was made to propagate IMNV in apparently healthy C6/36 subclone of Aedes albopictus cell line. The confirmatory assays such as RT-PCR, real-time PCR and bioassay revealed that C6/36 cells were found to be susceptible to IMNV and these cells could be used easily for isolation and propagation of IMNV. The results of real-time PCR assay showed that a lower CT value of 22.25 in IMNV-infected cells was obtained on 10day post-infection (d p.i.), whereas the higher CT value of 35.21 was obtained in IMNV-infected cells on 2d p.i. There is no significant difference between CT values of IMNV production in vitro using C6/36 cell line and in vivo using shrimp. The IMNV propagated in C6/36 cells is capable of infecting shrimp and caused 100% mortality in shrimp. Clinical signs observed in shrimp injected with IMNV propagated in C6/36 cell line were found to be similar to naturally infected shrimp.

Application of Brown Algae (Sargassum angustifolium) Extract for Improvement of Drought Tolerance in Canola (Brassica napus L.).

Background: Abiotic environmental stresses, especially drought stress, is one of the most important problems in arid and semi-arid regions. Like other major crops, Brassica napus is vulnerable to drought stress.Objective: The present study was conducted to evaluate efficacy of Sargassum angustifolium extract on mitigating adverse effects of drought stress on B. napus seedlings during vegetative growth under greenhouse conditions.Materials and Methods: Seedlings were periodically sprayed with the seaweed extract until they reached 7-leaf stage. Then water deficit stress was imposed and measurements were performed at morphological, biochemical and molecular levels on three phases: 80% field capacity for 20 days (Phase I), 60% field capacity for 20 days (Phase II) and 40% field capacity for 20 days (Phase III). Real-Time PCR assay was carried out to monitor the changes in expression of the genes involved in proline biosynthesis.Results: Morphological measurements revealed that seaweed treatment improved shoot height and dry weight compared to control (p<0.05). Biochemical analyses indicated that foliar application of seaweed extract significantly enhanced the photosynthetic pigments’ content, free radical scavenging and superoxide dismutase activity (p<0.05). Moreover, proline content was significantly increased in plant tissues treated with seaweed extract (p<0.05). The results of Real-Time PCR assay showed that the increase in proline content is due to enhanced expression of P5CS which is involved in biosynthesis of proline, and to decreased expression of PRODH which catalyzes proline degradation. Conclusions: Overall, the results obtained in this research suggest that application of S. angustifolium extract as a biostimulant is able to protect canola seedlings against deteriorating effects of drought stress.

Exploring Kawasaki disease-specific hub genes revealing a striking similarity of expression profile to bacterial infections using weighted gene co-expression network analysis (WGCNA) and co-expression modules identification tool (CEMiTool): An integrated bioinformatics and experimental study

Kawasaki disease (KD) has been declared a rare idiopathic condition for a long time. The children age less than five years, as the most susceptible group, are at risk of this disease.Since the cause of the disease is unknown, this study was designed to investigate the cause of KD. We applied metaDE and WGCNA packages in order to perform a meta-analysis and identify network modules of co-expressed genes, respectively, on three expression array datasets and also CEMiTool package to confirm detected modules by WGCNA. Using the Pearson correlation coefficient, the resemblance of KD to other symptomatic-similar diseases, including bacterial infections, viral infections, JIA (juvenile idiopathic arthritis), HSP (Henoch-Schönlein purpura), GAS (group A streptococcal), and HAdV (adenovirus) was accurately estimated. In addition to validation by more three expression array datasets, serum samples of 16 patients and eight control participants have undergone the Real-Time PCR assay so as to evaluate produced bioinformatic results. WGCNA showed 3840 differentially expressed genes (DEGs) in KD in comparison with other diseases accompanying resembling clinical manifestations. Through further bioinformatic analysis and validation, 42 out of DEGs were introduced as hub genes, which the results of Real-Time PCR assay subsequently attested to the majority of them. The DEGs possessed a remarkable commonality with those of bacterial conditions. According to our exhaustive results, the origin of KD has been revealed pertinent to bacterial infections. Another interesting finding in this study is introducing IVIG in combination with particular antibiotics as a novel therapeutic approach, which supported by a score of authentic research studies to overcome KD.

Overexpression of SMN2 Gene in Motoneuron-Like Cells Differentiated from Adipose-Derived Mesenchymal Stem Cells by Ponasterone A.

Cell therapy and stem cell transplantation strategies have provided potential therapeutic approaches for the treatment of neurological disorders. Adipose-derived mesenchymal stem cells (ADMSCs) are abundant adult stem cells with low immunogenicity, which can be used for allogeneic cell replacement therapies. Differentiation of ADMSCs into acetylcholine-secreting motoneurons (MNs) is a promising treatment for MN diseases, such as spinal muscular atrophy (SMA), which is associated with the level of SMN1 gene expression. The SMN2 gene plays an important role in MN disorders, as it can somewhat compensate for the lack of SMN1 expression in SMA patients. Although the differentiation potential of ADMSCs into MNs has been previously established, overexpression of SMN2 gene in a shorter period with a longer survival has yet to be elucidated. Ponasterone A (PNA), an ecdysteroid hormone activating the PI3K/Akt pathway, was studied as a new steroid to promote SMN2 overexpression in MNs differentiated from ADMSCs. After induction with retinoic acid, sonic hedgehog, forskolin, and PNA, MN phenotypes were differentiated from ADMSCs, and immunochemical staining, specific for β-tubulin, neuron-specific enolase, and choline acetyltransferase, was performed. Also, the results of real-time PCR assay indicated nestin, Pax6, Nkx2.2, Hb9, Olig2, and SMN2 expression in the differentiated cells. After 2weeks of treatment, cultures supplemented with PNA showed a longer survival and a 1.2-fold increase in the expression of SMN2 (an overall 5.6-fold increase; *P ≤ 0.05), as confirmed by the Western blot analysis. The PNA treatment increased the levels of ChAT, Isl1, Hb9, and Nkx2 expression in MN-like cells. Our findings highlight the role of PNA in the upregulation of SMN2 genes from MSC-derived MN-like cells, which may serve as a potential candidate in cellular therapy for SMA patients.

Application of a simple and high-throughput DNA extraction method to real-time PCR quantification of target plant-parasitic nematodes in nematode communities

Molecular techniques are prevailing in nematode identification and quantification, for which DNA extraction from nematodes is essential. However, the published DNA extraction methods are laborious and often require various expensive consumables and high-end equipment. In order to prepare DNA templates for a high-throughput real-time PCR assay, the present study modified a conventional DNA extraction method of Naklha et al. (2010) from nematode suspensions with ordinary lab equipment and achieved results and advantages comparable with two other conventional methods. The results of real-time PCR assays for quantifying Pratylenchus zeae, Tylenchorhynchus leviterminalis and Hoplolaimus sp. using the new protocol were highly correlated with those obtained by morphological counts and comparable to or more sensitive than those obtained by two conventional methods. Although the new protocol took over 100 min for DNA extraction, the manual processing took less than 10 min, i.e., half to one-fourth of the other methods. The running cost was less than half to one-tenth of the other methods.

Detection of Salmonella enterica subsp. enterica Serovar Cubana from Naturally Contaminated Chick Feed

Because some significant outbreaks of human salmonellosis have been traced to contaminated animal feed, the rapid and efficient detection of Salmonella in feed is essential. However, the current U.S. Food and Drug Administration Bacteriological Analytical Manual (BAM) culture method that uses lactose broth as a preenrichment medium has not reliably supported the results of real-time PCR assays for certain foods. We evaluated the BAM culture method and a quantitative real-time PCR (qPCR) assay using two preenrichment media, modified buffered peptone water and lactose broth, to detect Salmonella enterica subsp. enterica serovar Cubana in naturally contaminated chick feed. After 24 h of incubation, the qPCR method was as sensitive as the culture method when modified buffered peptone water was used as the preenrichment medium but less sensitive than culture when lactose broth was used. After 48 h of incubation, detection of Salmonella Cubana by qPCR and by culture in either preenrichment medium was equivalent. We also compared the performance of the traditional serotyping method, which uses pure cultures of Salmonella grown on blood agar, to two molecular serotyping methods. The serotyping method based on whole genome sequencing also requires pure cultures, but the PCR-based molecular serotyping method can be done directly with the enriched culture medium. The PCR-based molecular serotyping method provided simple and rapid detection and identification of Salmonella Cubana. However, whole genome sequencing allows accurate identification of many Salmonella serotypes and highlights variations in the genomes, even in tight genomic clusters. We also compared the genome of the chick feed isolate with 58 Salmonella Cubana strains in GenBank and found that the chick feed isolate was very closely related to an isolate from a foodborne outbreak involving alfalfa sprouts.

Pregnancy rates of beef cattle are not affected by Campylobacter fetus subsp. venerealis real-time PCR-positive breeding sires in New Zealand

AIMS: Campylobacter fetus subspecies venerealis (C. fetus venerealis) is the causal agent of bovine genital campylobacteriosis, a venereal disease that is asymptomatic in bulls but responsible for reproductive wastage in female cattle. In New Zealand, a commercial real-time PCR assay was introduced in 2007 to identify the DNA of this pathogen in preputial scrapings; however, concerns were raised about the specificity of the test following anecdotal reports of a high number of test-positive bulls with no apparent relationship to reproductive performance. The objective of this study, therefore, was to examine the association between real-time PCR assay results from beef breeding bulls and pregnancy rates in beef herds using these bulls.METHODS: Veterinarians from four veterinary practices selected beef cattle herds with relatively high and low pregnancy rates between December 2008 and February 2009. Preputial scrapings were collected from bulls used for mating in those herds. Samples were tested using the real-time PCR assay under consideration. Bivariable and multivariable analyses were used to assess the relationship between pregnancy rates in each mob (15-month-old heifers, 27-month-old heifers and mixed-age cows) and the percentage of real-time PCR-positive bulls in each mob.RESULTS: Sixty-four (28.8%) of 222 bulls tested positive, 130 (58.6%) tested negative, and 28 (12.6%) returned an inconclusive result to the real-time PCR assay. The percentage of bulls testing real-time PCR-positive in these mobs was not associated with pregnancy rates (p=0.757) after controlling for mob, average body condition score of cows, cow to bull ratio, length of the mating period, and farm.CONCLUSION: Real-time PCR assay results were not associated with pregnancy rates, suggesting that the specificity of the real-time PCR assay was too low to be used to reliably detect C. fetus venerealis. This study adds to a growing body of evidence indicating that C. fetus venerealis strains are either absent from, or present at clinically insignificant levels of endemicity among, beef breeding herds in New Zealand.CLINICAL SIGNIFICANCE: The real-time PCR assay that was assessed in this study should not be used for the detection of C. fetus venerealis in bulls or for investigations of low conception rates in cattle in New Zealand. During the course of this survey, sequencing analysis of an apparent C. fetus venerealis isolate from the intestines of a Friesian bull turned out to be Campylobacter hyointestinalis. As a consequence, this real-time PCR assay for C. fetus venerealis is no longer being offered by diagnostic laboratories in New Zealand.

Detection of influenza A viruses with a portable real-time PCR instrument

Timely identification of respiratory pathogens is essential for appropriate patient care and cohorting. In order to do rapid identification-technology near the patient we utilized the field-deployable RAZOR EX-thermocycler with a reverse transcription real-time PCR assay that detects all subtypes of influenza A virus. In addition, we developed a RT PCR assay for specific detection of influenza A(H1N1)pdm09 virus. These assays amplified segments of the matrix (M)- and the hemagglutinin (HA)-gene, respectively. Detection limits of the M-gene and the influenza A(H1N1)pdm09-specific HA-gene assays were 0.15 PFU and 8.8 PFU per reaction, respectively. With 18 influenza A viruses of different subtypes and influenza B, C, and 7 other respiratory viruses the RAZOR EX and standard real-time PCR assay results were in total agreement. From 104 clinical samples identical results were obtained by both PCR methods. Additional 21 clinical samples were tested under field conditions with the RAZOR EX instrument. Results were achieved in 90 min, including 45 min for sample preparation and they were in complete agreement with those obtained by standard real-time PCR under laboratory conditions. These methods enable highly sensitive and rapid on-site diagnostics to reliably identify patients infected with influenza A, including the influenza A(H1N1)pdm09-virus.

A rapid low-cost real-time PCR for the detection of klebsiella pneumonia carbapenemase genes

BackgroundKlebsiella pneumonia carbapenemases (KPCs) are able to hydrolyze the carbapenems, which cause many bacteria resistance to multiple classes of antibiotics, so the rapid dissemination of KPCs is worrisome. Laboratory identification of KPCs-harboring clinical isolates would be a key to limit the spread of the bacteria. This study would evaluate a rapid low-cost real-time PCR assay to detect KPCs.MethodsReal-time PCR assay based on SYBR GreenIwas designed to amplify a 106bp product of the blaKPC gene from the159 clinical Gram-negative isolates resistant to several classes of -lactam antibiotics through antimicrobial susceptibility testing. We confirmed the results of real-time PCR assay by the conventional PCR-sequencing. At the same time, KPCs of these clinical isolates were detected by the modified Hodge test (MHT). Then we compared the results of real-time PCR assay with those of MHT from the sensitivity and specificity. Moreover, we evaluated the sensitivity of the real-time PCR assay.ResultsThe sensitivity and specificity of the results of the real-time PCR assay compared with those of MHT was 29/29(100%) and 130/130(100%), respectively. The results of the real-time PCR and the MHT were strongly consistent (Exact Sig. (2-tailed) =1. 000; McNemar test). The real-time PCR detection limit was about 0.8cfu using clinical isolates.ConclusionThe real-time PCR assay could rapidly and accurately detect KPCs -harboring strains with high analytical sensitivity and specificity.

Irrigation effects on pds and bch genes expression of the Iranian Saffron

Saffron (Crocus sativus L.) is an indigenous and the most valuable and marginal plant in Iran. However, limited knowledge exists on its molecular biology. The importance of this plant is due to the color, flavor and medicinal properties of its red stigmas. Saffron stigmas contain a high amount of carotenoids such as crocetin and crocin. Two genes, bch and pds, have essential role in carotenoid production. In this study, the effect of four different irrigation regimes was evaluated on the expression of bch and pds genes. Semi-quantitative RTPCR showed no significant difference in the expression levels of genes of interest related to the internal standard (18S rRNA). Results of Real-Time PCR assays showed that the expression of bch and pds genes were affected by irrigation treatments as their expression decreased in irrigated plants in comparison to non irrigated ones, exept for one irrigation treatment (one irrigation in September) where the pds gene expression showed higher level. However, the expression profile of the genes was almost the same in all treatments. The comparison between results of two techniques indicated that the Real-Time PCR is more accurate for determination of the level of transcript in the Iranian saffron. It was interesting that by decreasing of irrigation, the expression level of these two genes increased indicating that abiotic stress and drought can affect on the gene expression relating to the saffron color.

Detection of Yersinia pestis using real-time PCR in patients with suspected bubonic plague

Yersinia (Y.) pestis, the causative agent of plague, is endemic in natural foci of Asia, Africa, and America. Real-time PCR assays have been described as rapid diagnostic tools, but so far none has been validated for its clinical use. In a retrospective clinical study we evaluated three real-time PCR assays in two different assay formats, 5′-nuclease and hybridization probes assays. Lymph node aspirates from 149 patients from Madagascar with the clinical diagnosis of bubonic plague were investigated for the detection of Y. pestis DNA. Results of real-time PCR assays targeting the virulence plasmids pPCP1 ( pla gene), and pMT1 ( caf1, Ymt genes) were compared with an F1-antigen immunochromatographic test (ICT) and cultivation of the organism. Out of the 149 samples an infection with Y. pestis was confirmed by culture in 47 patients while ICT was positive in 88 including all culture proven cases. The best real-time PCR assay was the 5′-nuclease assay targeting pla which was positive in 120 cases. In conclusion, the 5′-nuclease assay targeting pla can be recommended as diagnostic tool for establishing a presumptive diagnosis when bubonic plague is clinically suspected.

Culture-Negative Childhood Empyema Is Usually Due to Penicillin-Sensitive Streptococcus pneumoniae Capsular Serotype 1

Two studies in the United States have shown that isolates of Streptococcus pneumoniae from children with empyema are more likely to be penicillin sensitive and of capsular serotype 1 than those from uncomplicated pneumococcal pneumonia (1, 4). In the United Kingdom, childhood empyema is becoming more common (S. D. Playfor, A. R. Smyth, and R. J. Stewart, Letter, Thorax 52:932, 1977; J. H. M. Rees, D. A. Spencer, D. Parikh, and P. Weller, Letter, Lancet 349:402, 1997). Many cases have no definitive etiology, despite attempts at noncultural diagnosis by antigen detection, and are most likely due to previous antibiotic therapy. It is uncertain, therefore, if these culture-negative cases of childhood empyema are from the same population as the S. pneumoniae culture-positive ones or whether they are a separate and different problem. We have examined pleural aspirates from 43 children (aged 6 months to 17 years; mean age, 6.2 years; median age, 6 years; 30 of whom were male) presenting with empyema thoracis over a 4-year period to the Freeman Hospital in Newcastle upon Tyne, a tertiary referral center for the North East of England. Routine culture and a latex agglutination test for pneumococcal antigen (SLIDEX; BioMerieux, Marcy l'Etoile, France) were performed with these specimens, after which the remaining material was digested with dithiothreitol. A 1-ml aliquot of the digest was centrifuged at 10,000 × g for 10 min, and the pellet was washed twice in phosphate-buffered saline (PBS). The pellet was resuspended in 12.5% Chelex-100 resin (Bio-Rad Laboratories Ltd.) and heated at 56°C for 30 min and then at 100°C for 15 min. The supernatant was then tested for the presence of S. pneumoniae DNA by a previously described real-time PCR assay targeting the pneumolysin gene (ply) (A. M. Kearns, R. Freeman, O. M. Murphy, P. R. Seiders, M. Steward, and J. Wheeler, Letter, J. Clin. Microbiol. 37:3434, 1999). Positive samples underwent a second real-time PCR assay targeting a conserved sequence of the PBP2B gene (pbp2b) (3) found in all strains of penicillin-susceptible S. pneumoniae (PSSP) for which the MIC of penicillin was <0.06 mg/liter. Finally, whenever sample volumes permitted, 250 μl of the digested sample was mixed with 750 μl of PBS and heated to 100°C for 10 min and then centrifuged for 2 min (21,000 × g). The supernatant was used in solid-phase indirect sandwich enzyme-linked immunosorbent assays (ELISAs) for the detection of type-specific pneumococcal capsular polysaccharides, with monoclonal antibodies to types 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F donated by Wyeth Vaccines Research, West Henrietta, N.Y. Two samples were culture positive. One of them, for which all tests for S. pneumoniae were negative, yielded Staphylococcus aureus. The other grew scanty Staphylococcus epidermidis, which was regarded as a skin contaminant. All pbp2b assays indicated “penicillin sensitivity.” Other results are found in Table ​Table11. TABLE 1. Results of real-time PCR assays for pneumococcal DNA and ELISAs for 13 pneumococcal capsular serotype antigens on 43 pleural aspirates from children with empyema We found evidence of S. pneumoniae infection in over 70% of culture-negative empyema fluids. The infecting strains were likely to have been highly penicillin sensitive, and at least 60% were of capsular serotype 1, which, in the United Kingdom, as in the United States, is not the commonest serotype in invasive pneumococcal disease (2). We conclude that cases of culture-negative childhood empyema presenting at tertiary referral centers are not an etiologically distinct group, that childhood pneumococcal vaccines must include capsular serotype 1 antigen, and that early detection of S. pneumoniae capsular serotype 1 antigen in cases of childhood pneumonia may aid management and predict complications, especially empyema.