Detection of Bacillus cereus as a causative agent of emetic food poisoning by an unconventional culture procedure

Bacillus cereus is known to cause two types of food poisoning: emetic and diarrhoeal. Both diseases are usually self-limiting; however, severe cases have been reported, presenting with acute liver failure and encephalopathy, including rarely fatal cases of vomiting. Clinical laboratories do not routinely test for B. cereus in patients with gastrointestinal disease. Therefore, B. cereus causing food poisoning goes undetected. We report a successful isolation of emetic B. cereus from a patient with food poisoning who presented with severe vomiting, fulminant hepatic failure, and acute encephalopathy, by a non-conventional method. Initially, stool specimens from the patients were routinely cultured to identify the causative organisms of food poisoning. No foodborne pathogens were detected in this study. In contrast, additional clinical and epidemiological information strongly suggested food poisoning by emetic B. cereus. Consequently, we allowed Drigalski agar medium smeared with patient stool specimens to stand at room temperature (approximately 25 °C) for 9 days. After 9 days, mixed bacteria grown on the medium were inoculated onto mannitol egg yolk polymyxin (MYP) agar plates, a selective medium for B. cereus. Typical colonies of B. cereus developed on MYP agar plates. The isolated B. cereus had a cereulide-producing genetic locus (ces) gene encoding the emetic toxin cereulide. The method used in this case study was unique. This method is easy to apply after obtaining an additional clinical and epidemiological information, and this method will improve the diagnostic rate of severe B. cereus food poisoning. This will contribute to the advancement of therapeutics in the future.     

Real-time PCR using SYBR Green for the detection of Shigella spp. in food and stool samples

Shigella spp are exquisitely fastidious Gram negative organisms that frequently get missed in the detection by traditional culture methods. For this reason, this work has adapted a classical PCR for detection of Shigella in food and stool specimens to real-time PCR using the SYBR Green format. This method follows a melting curve analysis to be more rapid and provide both qualitative and quantitative data about the targeted pathogen.A total of 117 stool samples with diarrhea and 102 food samples were analyzed in Public Health Regional Laboratory of Nabeul by traditional culture methods and real-time PCR. To validate the real-time PCR assay, an experiment was conducted with both spiked and naturally contaminated stool samples. All Shigella strains tested were ipaH positive and all non-Shigella strains yielded no amplification products. The melting temperature (T_m = 81.5 ± 0.5 °C) was consistently specific for the amplicon. Correlation coefficients of standard curves constructed using the quantification cycle (C_q) versus copy numbers of Shigella showed good linearity (R² = 0.995; slope = 2.952) and the minimum level of detection was 1.5 × 10³ CFU/g feces. All food samples analyzed were negative for Shigella by standard culture methods, whereas ipaH was detected in 8.8% culture negative food products. Moreover, the ipaH specific PCR system increased the detection rate over that by culture alone from 1.7% to 11.1% among patients with diarrhea.The data presented here shows that the SYBR Green I was suitable for use in the real-time PCR assay, which provided a specific, sensitive and efficient method for the detection and quantification of Shigella spp in food and stool samples.     

Differentiation of Variola major and Variola minor variants by MGB-Eclipse probe melt curves and genotyping analysis

Smallpox, caused by the Variola major virus, is considered to be one of the most lethal of all potential biological weapons and has far-reaching consequences. Real-time polymerase chain reaction (PCR) assays are available as a reliable diagnostic tool to detect members of the genus Orthopoxvirus. In addition real-time PCR assays specific for the variola virus have been developed that distinguish it from other orthopoxviruses. However, a positive identification of variola spp. does not classify the virus as the one that causes smallpox (V. major) or as the variant (Variola minor) that causes a much less severe form of the disease. This study reports the development of a real-time PCR minor groove binder (MGB)-Eclipse probe assay utilizing a sequence within the variola B9R/B10R gene complex that reliably differentiates V. major from V. minor by specific probe melting temperatures (T_ms) and genotyping analysis. The MGB-Eclipse probe assay is an important step beyond the standard TaqMan-MGB assay and we feel this is a significant addition to our current variola species identification algorithm with TaqMan-MGB assays that target the B9R and B10R genes. The probe T_ms for V. major and V. minor were 62.71 (±0.05) and 53.97 (±0.44) °C, respectively (P = <0.001). We also used the identical sequence to develop a TaqMan^®-MGB assay that specifically detected V. minor but not V. major variants by qualitative analysis.     

蜡样芽胞杆菌快速检测及其毒力基因筛选

目的筛选蜡样芽胞杆菌鉴定基因和快速检测毒力基因。方法选择分离自食品和土壤的代表性蜡样芽胞杆菌共329株,聚合酶链式反应（PCR）方法检测gyrB和groEL基因的种特异性,检测毒力基因在菌株中的分布特征。 基于检测结果,用多重PCR检测方案快速检测蜡样芽胞杆菌鉴定基因及其毒力因子。结果在蜡样芽胞杆菌及其近缘芽胞杆菌中,除1株苏云金芽胞杆菌扩增阳性外,gyrB基因具有蜡样芽胞杆菌种特异性;而groEL基因在4种芽胞杆菌中均有扩增。 6种毒力基因nheA、entFM、bceT、hblC、cytK和ces的携带率分别为84.19%、79.64%、49.24%、47.72%、47.11%和1.52%。 选择nheA、hblC、entFM、ces、cytK和gyrB用于快速鉴定蜡样芽胞杆菌及其毒力基因,获得了双重PCR扩增体系（gyrB和cytK）与4重PCR扩增体系（nheA、hblC、entFM和ces）的最佳检测方案。结论筛选的蜡样芽胞杆菌鉴定基因和毒力基因能够全面、特异、简便、高效地检测蜡样芽胞杆菌,可为食品安全检测及快速诊断提供依据,在实际检验工作中具有良好的应用前景。     

Identification of distinct ciprofloxacin susceptibility in Acinetobacter spp. by detection of the gyrA gene mutation using real-time PCR

Ciprofloxacin-resistant (CipR) Acinetobacter spp. was associated with a mutation in quinolone resistance-determining region (QRDR) of gyrA gene from Ser83 to Leu83. A total of 54 Acinetobacter baumannii, 11 A. genospecies 3, and 17 A. genospecies 13TU clinical isolates were determined for ciprofloxacin susceptibility and their QRDR sequenced. Most of A. baumannii were CipR and had a mutated QRDR of gyrA gene, and all A. genospecies 3 and 13TU isolates were ciprofloxacin-susceptible (CipS) and had a wild-type QRDR of gyrA gene. A real-time PCR assay was developed to rapidly differentiate the CipR and CipS Acinetobacter spp. This assay was based on probe hybridization followed by melting temperature (T_m) analysis to discriminate the QRDR of gyrA gene. All CipR strains had a T_m at 47 °C, and most of CipS strains had a higher T_m at 51.5 °C. Four CipS A. genospecies 3 isolates with an A base at 264 nt of the gyrA gene had a T_m at 49.5 °C. The assay can rapidly and accurately identify the mutated QRDR of gyrA gene in CipR Acinetobacter spp., and potentially increase the rate of the appropriate therapy for Acinetobacter infections.     

Multiplex PCR for the detection and differentiation of Vibrio parahaemolyticus strains using the groEL,tdh and trh genes

Vibrio parahaemolyticus is a significant cause of human gastrointestinal disorders worldwide, transmitted primarily by ingestion of raw or undercooked contaminated seafood. In this study, a multiplex PCR assay for the detection and differentiation of V. parahaemolyticus strains was developed using primer sets for a species-specific marker, groEL, and two virulence markers, tdh and trh.Multiplex PCR conditions were standardised, and extracted genomic DNA of 70 V. parahaemolyticus strains was used for identification. The sensitivity and efficacy of this method were validated using artificially inoculated shellfish and seawater. The expected sizes of amplicons were 510 bp, 382 bp, and 171 bp for groEL, tdh and trh, respectively. PCR products were sufficiently different in size, and the detection limits of the multiplex PCR for groEL, tdh and trh were each 200 pg DNA. Specific detection and differentiation of virulent from non-virulent strains in shellfish homogenates and seawater was also possible after artificial inoculation with various V. parahaemolyticus strains.This newly developed multiplex PCR is a rapid assay for detection and differentiation of pathogenic V. parahaemolyticus strains, and could be used to prevent disease outbreaks and protect public health by helping the seafood industry maintain a safe shellfish supply.     

Establishment of a duplex SYBR green I-based real-time polymerase chain reaction assay for the rapid detection of canine circovirus and canine astrovirus

The similar clinical characteristics of canine circovirus (CaCV) and canine astrovirus (CaAstV) infections and high frequency of co-infection make diagnosis difficult. In this study, a duplex SYBR Green I-based real-time polymerase chain reaction (PCR) assay was established for the rapid, simultaneous detection of CaCV and CaAstV. Two pairs of specific primers were designed based on the Rep gene of CaCV and the Cap gene of CaAstV. By using the real-time PCR assay method, the two viruses can be distinguished by the difference in melting temperatures, 79 °C and 86 °C for CaCV and CaAstV, respectively. This assay had high specificity, showing no cross-reaction with other common canine viruses, as well as high sensitivity, with minimum detection limits of 9.25 × 10¹ copies/μL and 6.15 × 10¹ copies/μL for CaCV and CaAstV, respectively. Based on the mean coefficient of variation, the method had good reproducibility and reliability. In a clinical test of 57 fecal samples, the rates of positive detection by real-time PCR were 14.04% (8/57) and 12.28% (7/57) for CaCV and CaAstV, respectively, and the rate of co-infection was 8.77% (5/57). In conclusion, the newly established duplex SYBR Green I-based real-time PCR assay is sensitive, specific, reliable, and rapid and is an effective tool for the detection of co-infections with CaCV and CaAstV.     

Simultaneous differentiation and diagnosis of goose parvovirus and astrovirus in clinical samples with duplex SYBR Green I real-time PCR

Two pairs of primers were designed to bind conserved genomic regions of goose parvovirus (GPV) and goose astrovirus (GAstV) to establish a simple, sensitive, and highly specific duplex quantitative PCR (qPCR) method to simultaneously detect the two viruses. The duplex qPCR can distinguish GPV (melting point: 82.1 °C) and GAstV (melting point: 79.8 °C) by the peaks of their individual melting curves. Mixed testing with other waterfowl viruses produced no nonspecific peaks. The established standard curves showed good linear relationships (R² > 0.997) and the limits of detection (LOD) for GPV and GAstV were 5.74 × 10¹ and 6.58 × 10¹ copies/μL, respectively. Both intra- and inter-assay coefficients of variation were <2%, indicating that the method has good repeatability. Twenty tissue samples from diseased geese were examined with the duplex qPCR assay and conventional PCR. Duplex qPCR showed positive rates of 25% for GPV and 45% for GAstV, and the positive rate for GPV and GAstV coinfection was 15%, slightly higher than the results for conventional PCR. These results indicated that this duplex qPCR method is highly sensitive, specific, and reproducible, and is suitable for epidemiological studies to effectively control the transmission of GPV and GAstV.     

High resolution melting-curve (HRM) analysis for the diagnosis of cryptosporidiosis in humans

Cryptosporidiosis of humans is an intestinal disease caused predominantly by infection with Cryptosporidium hominis or C. parvum. This disease is transmitted mainly via the faecal-oral route (water or food) and has major socioeconomic impact globally. The diagnosis and genetic characterization of the main species and population variants (also called “genotypes” and “subgenotypes”) of Cryptosporidium infecting humans is central to the prevention, surveillance and control of cryptosporidiosis, particularly as there is presently no cost effective anti-cryptosporidial chemotherapeutic regimen or vaccine available. In the present study, we established a polymerase chain reaction (PCR)-coupled high resolution melting-curve (HRM) analysis method, utilizing the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA as the genetic marker, for the diagnosis of Cryptosporidium hominis, C. parvum or C. meleagridis infection. An evaluation of the method revealed intra- and inter-assay variabilities of <1.5 and 3.5%, respectively. Cryptosporidium hominis, C. parvum and C. meleagridis were detected in 97, 44 and 2, respectively, of the 143 Cryptosporidium oocyst DNA samples originating from Australians with clinical cryptosporidiosis. The melting profiles characterized by peaks of 72.47 ± 0.33 °C and 74.19 ± 0.45 °C (profile 1), 72.17 ± 0.32 °C (profile 2) and 73.33 ± 0.03 °C (profile 3) genetically identified as C. hominis, C. parvum and C. meleagridis, respectively. In conclusion, PCR-coupled melting analysis of ITS-2 achieved the diagnosis of Cryptosporidium hominis, C. parvum or C. meleagridis infection. This approach is well suited for the rapid screening of large numbers of Cryptosporidium oocyst DNA samples and, although qualitative, is significantly less time-consuming to carry out than electrophoretic analysis and has the added advantage of data storage and analysis capabilities in silico. This method provides a useful tool for investigating the epidemiology and outbreaks of cryptosporidiosis, and could be applicable to species of Cryptosporidium other than those investigated herein.     

Multiplex real-time PCR assay for detection of pathogenic Vibrio parahaemolyticus strains

Foodborne disease caused by pathogenic Vibrio parahaemolyticus has become a serious public health problem in many countries. Rapid diagnosis and the identification of pathogenic V. parahaemolyticus are very important in the context of public health. In this study, an EvaGreen-based multiplex real-time PCR assay was established for the detection of pathogenic V. parahaemolyticus. This assay targeted three genetic markers of V. parahaemolyticus (species-specific gene toxR and virulence genes tdh and trh). The assay could unambiguously identify pathogenic V. parahaemolyticus with a minimum detection limit of 1.4 pg genomic DNA per reaction (concentration giving a positive multiplex real-time PCR result in 95% of samples). The specificity of the assay was evaluated using 72 strains of V. parahaemolyticus and other bacteria. A validation of the assay with clinical samples confirmed its sensitivity and specificity. Our data suggest the newly established multiplex real-time PCR assay is practical, cost-effective, specific, sensitive and capable of high-throughput detection of pathogenic V. parahaemolyticus.     

Motion characteristics of the lower lumbar spine in individuals with different pelvic incidence: An in vivo biomechanical study

BackgroundPelvic incidence is the quantification of the pelvis anatomical shape which has significant effect on the occurrence of various lumbar degenerative diseases. The aim of this study was to measure the in vivo dynamic motion characteristics of the lower lumbar spine in people with different pelvic incidence.MethodsA total of 55 volunteers were included in the study. The participants were devided into 3 groups (A: pelvic incidence≤40°, B: 40° < pelvic incidence <60° and C: pelvic incidence ≥60°). The L3–S1 vertebrae of each subject was MRI scanned to construct 3D models. The lumbar spine was then imaged using a dual fluoroscopic imaging system as the subject performed physiological position. The 3D vertebral models and the fluoroscopic images were used to reproduce the in vivo vertebral positions along the motion path. The relative translations and rotations of each motion segment were analyzed.FindingsAt the L5-S1 segment, the primary ranges of motion for left-right axial rotation and flexion-extension of the patients with large pelvic incidence (3.28° ± 0.79°, 7.56° ± 1.81°) were significantly larger than normal pelvic incidence (2.61° ± 1.01°, 6.57° ± 2.18°) and small pelvic incidence (2.00° ± 0.60°, 5.83° ± 1.67°).InterpretationThe anatomic variable pelvic incidence is associated with the ranges of motion in lower lumbar vertebrae, especially in the L4–5 and L5-S1 segments.     

Multiplex qPCR assay for assessment of reference gene expression stability in rat tissues/samples

RT-qPCR requires an adequate choice of stably expressed reference genes for accurate normalization of mRNA expression. However, testing a panel of reference genes is often time-consuming and expensive. In this work, we aimed to develop a set of multiplex real-time PCR assays for RT-qPCR analysis of commonly used housekeeping genes in laboratory rats. Using Hydrolysis probe (TaqMan®) technology, we have designed and optimized three triplex qPCR assays (Actb + Gapdh + B2m; Rpl13a + Sdha + Ppia; Hprt1+Pgk1+Ywhaz) demonstrating optimal PCR amplification efficiencies (from 94.7 to 100.5%) and repeatability. Novel assays allow expression analysis of 9 reference genes in 3 reactions making possible a more time-efficient choice of reference genes in RT-qPCR experiments in Wistar rats in comparison with widespread singleplex assays.     

A study of knee anterior cruciate ligament biomechanics with respect to energy and relaxation

BackgroundThis research aimed to study the biomechanical properties of sheep tendon under torsion and the tendon energy absorption performance with an externally imposed initial force.MethodsTendons of nine healthy knees of sheep were investigated. In both tests, we investigated energy and relaxation at rotations of 0°, 90°, 180°, and 360°. For both tensile force and tensile displacement at a sampling period of 100 milliseconds, the maximum value of 89 N was selected as the maximum tension state for 600 s of relaxation duration for testing relaxation, and analysed of the average force of the last 30 s.FindingsThe difference of energy levels of the tendons are significant between twisted groups (180° and 360°) and untwisted group (0°) (P < 0.05); The relaxation force decreases significantly with twisted groups (90°,180°, and 360°) and untwists group (0°) (P < 0.05). The nine-group tendons show no significant difference at torsion 90° and 180° (P = 0.466). Peak force test shows significant differences between the twisted groups (90°,180°, and 360°) and untwisted group (0°) (P < 0.05).InterpretationThe torsion tendon has lower energy absorption and relaxation than the untwisted counterparts; thus, it may be more prone to damage. These results are useful for providing guidance on anterior cruciate ligament reconstruction.     

Controlled multiplex PCR of enterotoxigenicClostridium perfringensstrains in food samples

A controlled multiplex polymerase chain reaction (PCR) for the detection ofClostridium(C.)perfringensenterotoxin gene (cpe) was established and compared with anin vitroantigenic detection method. ThecpePCR and the classical method of electric immunodiffusion gave identical results. The predicted specific amplicon of thecpegene was generated from all of the tested enterotoxigenicC. perfringensstrains. In contrast, cultures of any otherClostridiumspecies tested by PCR were negative (100% sensitivity, 100% specificity). Addition of an alphatoxin (plc) gene specific PCR as an in process control reaction was performed in order to prevent false negative PCR results. The PCR detection limit was 0·5 ng of genomicC. perfringensDNA per ml of bouillon culture. By contaminating minced meat withC. perfringensreference strains, the multiplex PCR was established as a tool for routine diagnostic laboratories. The detection limit was approximately 3·0×10⁵C. perfringenscells per gram meat. The results demonstrate the multiplex PCR as an easy, specific, sensitive and time saving diagnostic procedure. Application of this improved method should enhance the knowledge concerning epidemiological aspects of food borne diseases caused by enterotoxigenicC. perfringens.     

A duplex SYBR Green I-based real-time RT-PCR assay for the simultaneous detection and differentiation of Massachusetts and non-Massachusetts serotypes of infectious bronchitis virus

Infectious bronchitis is a highly contagious viral disease of poultry caused by infectious bronchitis virus (IBV) and is considered one of the most economically important viral diseases of chickens. Control of IBV has been attempted using live attenuated and inactivated vaccines. Live attenuated vaccines of the Massachusetts (Mass.) serotype are the most commonly used for this purpose. Due to the continuous emergence of new variants of the infectious bronchitis virus, the identification of the type of IBV causing an outbreak in commercial poultry is important in the selection of the appropriate vaccine(s) capable of inducing a protective immune response. The present work was aimed at developing and evaluating a duplex SYBR Green I-based real-time RT-PCR (rRT-PCR) assay for the simultaneous detection and differentiation of Mass. and non-Mass. serotypes of IBV. The duplex rRT-PCR yielded curves of amplification with two specific melting curves (Tm1 = 83 °C ± 0.5 °C and Tm2 = 87 °C ± 0.5 °C) and only one specific melting peak (Tm = 87 °C ± 0.5 °C) when the IBV Mass. serotype and IBV non-Mass. serotype strains were evaluated, respectively. The detection limit of the assay was 8.2 gene copies/μL based on in vitro transcribed RNA and 0.1 EID₅₀/mL. The assay was able to detect all the IBV strains assessed and discriminated well among the IBV Mass. and the IBV non-Mass. serotypes strains. In addition, amplification curves were not obtained with any of the other viruses tested. From the 300 field samples tested, the duplex rRT-PCR yielded a total of 80 samples that were positive for IBV (26.67%), 73 samples identified as the IBV Mass. serotype and seven samples as identified as the IBV non-Mass. serotype. A comparison of the performance of test as assessed with field samples revealed that the duplex rRT-PCR detected a higher number of IBV-positive samples than when conventional RT-PCR or virus isolation tests were used. The duplex rRT-PCR presented here is a useful tool for the rapid identification of outbreaks and for surveillance programmes during IB-suspected cases, particularly in countries with a vaccination control programme.     

Electrophysiological Changes Correlated with Temperature Increases Induced by High-Intensity Focused Ultrasound Ablation

To gain better understanding of the detailed mechanisms of high-intensity focused ultrasound (HIFU) ablation for cardiac arrhythmias, we investigated how the cellular electrophysiological (EP) changes were correlated with temperature increases and thermal dose (cumulative equivalent minutes [CEM₄₃]) during HIFU application using Langendorff-perfused rabbit hearts. Employing voltage-sensitive dye di-4-ANEPPS, we measured the EP and temperature during HIFU using simultaneous optical mapping and infrared imaging. Both action potential amplitude (APA) and action potential duration at 50% repolarization (APD₅₀) decreased with temperature increases, and APD₅₀ was more thermally sensitive than APA. EP and tissue changes were irreversible when HIFU-induced temperature increased above 52.3 ± 1.4°C and log₁₀(CEM₄₃) above 2.16 ± 0.51 (n = 5), but were reversible when temperature was below 50.1 ± 0.8°C and log₁₀(CEM₄₃) below –0.9 ± 0.3 (n = 9). EP and temperature/thermal dose changes were spatially correlated with HIFU-induced tissue necrosis surrounded by a transition zone.     

The immediate effect of IASTM vs. Vibration vs. Light Hand Massage on knee angle repositioning accuracy and hamstrings flexibility: A pilot study

IntroductionThe effectiveness of novel soft-tissue interventions relative to traditional ones requires further exploration. The purpose of this pilot study was to evaluate the immediate effect of Instrument Assisted Soft Tissue Mobilization (IASTM) compared to Vibration Massage or Light Hand Massage on hamstrings’ flexibility and knee proprioception.Methods16 healthy non-injured male participants (mean age 23.7 years, height 1.80 cms and body mass 77.7 kg) were randomly assigned to the following interventions: (a) 5min IASTM, (b) 5min Vibration Massage and (c) 8min Light Hand-Massage, sequentially delivered to all participants with an in-between 1-week time interval. A single application of each intervention was given over the hamstrings of their dominant leg (repeated measures under 3 different experimental conditions). An active knee angle reproduction proprioception test and the back-saver sit and reach flexibility test were performed before and immediately after each intervention. Reliability of outcomes was also assessed.ResultsReliability for flexibility (ICC_3,1 = 0.97–0.99/SEM = 0.83–1.52 cm) and proprioception (ICC_3,1 = 0.83–0.88/SEM = 1.63–2.02°) was very good. For flexibility, statistically significant immediate improvement (p < 0.001) was noted in all 3 groups (1.61–3.23 cm), with no between-group differences. For proprioception, improvement in the IASTM (2.12°), Vibration Massage (0.32°) and Light Hand-Massage (1.17°) conditions was not statistically significant; no between-group differences were also evident.ConclusionsOur findings indicate that muscle flexibility was positively influenced immediately after a single intervention of IASTM, Vibration Massage or Light Hand Massage. Proprioception changes were not statistically significant either within or between groups. Further evaluation of those interventions in a larger population with hamstrings pathology is required.     

A repA-based ELISA for discriminating cattle vaccinated with Brucella suis 2 from those naturally infected with Brucella abortus and Brucella melitensis

The commonest ways of diagnosing brucellosis in animals include the Rose-Bengal plate agglutination test, the buffered plate agglutination test (BPA), the slide agglutination test, the complement fixation test, and the indirect enzyme linked immunosorbent assay (I-ELISA). However, these methods cannot discriminate the Brucella vaccine strain (Brucella suis strain 2; B. suis S2) from naturally acquired virulent strains. Of the six common Brucella species, Brucella melitensis, Brucella abortus, and B. suis are the commonest species occurring in China. To develop an ELISA assay that can differentiate between cows inoculated with B. suis S2 and naturally infected with B. abortus and B. melitensis, genomic sequences from six Brucella spp. (B. melitensis, B. abortus, B. suis, Brucella canis, Brucella neotomae and Brucella ovis) were compared using Basic Local Alignment Search Tool software. One particular gene, the repA-related gene, was found to be a marker that can differentiate B. suis from B. abortus and B. melitensis. The repA-related gene of B. suis was PCR amplified and subcloned into the pET-32a vector. Expressed repA-related protein was purified and used as an antigen. The repA-based ELISA was optimized and used as specific tests. In the present study, serum from animals inoculated with the B. suis S2 vaccine strain had positive repA-based ELISA results. In contrast, the test-positive reference sera against B. abortus and B. melitensis had negative repA-based ELISA results. The concordance rate between B. abortus antibody-negative (based on the repA-based ELISA) and the Brucella gene-positive (based on the ‘Bruce ladder’ multiplex PCR) was 100%. Therefore, the findings suggest that the repA-based ELISA is a useful tool for differentiating cows vaccinated with the B. suis S2 and naturally infected with B. abortus and B. melitensis.     

Multiplex real-time SYBR Green I PCR assays for simultaneous detection of 15 common enteric pathogens in stool samples

Diarrheal diseases account for more than 50% of foodborne diseases worldwide, the majority of which occur in infants and young children. The traditional bacterial detection method is complex and time-consuming; therefore, it is necessary to establish a rapid and convenient detection method that can detect multiple pathogens simultaneously. In this study, we developed a set of five multiplex real-time SYBR Green I PCR assays to simultaneously detect 15 common enteric pathogens based on the Homo-Tag Assisted Non-Dimer system. These assays effectively reduced primer-dimer formation and improved the stability, uniformity, and amplification efficiency of multiplex PCR. The detection limit of the multiplex SYBR Green I PCR system was approximately 10⁴–10⁶ CFU/mL for stool specimens. Furthermore, we vitrified heat-unstable components on the cap of a reaction tube, showing that Taq DNA polymerase, dNTPs, primers, and SYBR Green I remained stable at 25 °C. In summary, we developed multiplex SYBR Green I PCR assays that can simultaneously detect 15 enteric pathogens. This method is comprehensive, rapid, inexpensive, accurate, and simple and displays high specificity.     

Rapid genotyping of HLA-B27 among Korean population by real-time PCR melting curve analysis

BackgroundReal-time PCR and melting curve analysis is the relatively recent method for HLA-B27 genotyping, which has advantages of being simple and rapid.MethodsThe accuracy of melting curve analysis for HLA-B27 was assessed in 153 clinical samples and 52 DNA samples from International Histocompatibility Workshop (IHW) cell lines, with sequence-based typing (SBT) as the reference method. We predicted melting reaction for various HLA-B27 subtypes using simulation software.ResultsFor clinical samples, 53 HLA-B27-positive and 100 negative results by melting curve analysis were confirmed by completely concordant SBT results. The B*27:05 allele was found in 50 patients, and the B*27:04 allele in 3 patients. Among 62 known alleles, 21 alleles had differences in the target sequence, including 10 alleles having mismatches in the primer binding site. In these alleles, differences in melting points (T_m) were predicted to be ≤ 1.2 °C. The predicted results were obtained when IHW samples were tested, which revealed slight lower T_m for B*27:06 and negative results for B*27:07.ConclusionsGenotyping of HLA-B27 by melting curve analysis was fast and reliable for routine laboratory testing for frequent alleles. In silico melting simulations provided useful information about the utility and limitation of this method for diverse HLA-B27 alleles.