A Novel Isothermal Assay of Borrelia burgdorferi by Recombinase Polymerase Amplification with Lateral Flow Detection

A novel isothermal detection for recombinase polymerase amplification with lateral flow (LF-RPA) was established for Borrelia burgdorferi (B. burgdorferi) detection in this study. This assay with high sensitivity and specificity can get a visible result without any additional equipment in 30 min. We designed a pair of primers according to recA gene of B. burgdorferi strains and a methodology evaluation was performed. The results showed that the RPA assay based on the recA gene was successfully applied in B. burgdorferi detection, and its specific amplification was only achieved from the genomic DNA of B. burgdorferi. The detection limit of the new assay was about 25 copies of the B. burgdorferi genomic DNA. Twenty Lyme borreliosis patients’ serum samples were detected by LF-RPA assay, real-time qPCR and nested-PCR. Results showed the LF-RPA assay is more effective than nested-PCR for its shorter reaction time and considerably higher detection rate. This method is of great value in clinical rapid detection for Lyme borreliosis. Using the RPA assay might be a megatrend for DNA detection in clinics and endemic regions.     

Isolation and characterization of Listeria monocytogenes in Chinese food obtained from the central area of China

The objective of this study was to investigate the prevalence and characteristics of Listeria monocytogenes isolated from Chinese food, including frozen dumplings, flavored raw meat, roasted meat, braised meat, and a cold vegetable dish with sauce. A total of 900 food samples were collected from supermarkets, open-air markets, and delicatessens in three large cities in the central area of China to examine the presence of L. monocytogenes; 21 (2.3%) of the samples were positive for this pathogen. Among the different samples, braised meat showed the highest L. monocytogenes detection rate (4.4%). Samples obtained from delicatessens showed a much higher L. monocytogenes contamination rate (8.3%) than those from open-air markets (6.7%) or supermarkets (0%). Multilocus sequence typing (MLST) analysis indicated that the 21 bacterial isolates belonged to 12 ST subgroups. ST5 was the largest and contained 7 isolates (33.3%); it was followed by ST474, ST121 and ST9 (each containing 2 isolates [10.5%]). Antibiotic susceptibility analysis showed that the 21 L. monocytogenes isolates were thoroughly resistant to cefoxitin but highly susceptible to doxycycline and ciprofloxacin. The presence of 10 virulence genes was evaluated by PCR, which showed that inlA, inlC, inlJ, prfA, hlyA, and plcB were present in all isolates and that inlB, actA, plcA and iap were present in 71.4–90.5% of the isolates. This study provides a useful reference for risk assessment and control of L. monocytogenes contamination in Chinese food and for the treatment of clinical listeriosis.     

火腿肠加工过程中微生物风险研究

目的开展火腿肠加工过程中微生物污染风险研究,掌握卫生指示菌、主要食源性致病菌的分布特征和污染途径,为火腿肠加工过程中微生物污染风险控制提供依据。方法 2015—2017年对4家企业的712份产品相关样品(原辅料、中间产品和终产品)和环境样品(包括生产用水、空气沉降菌、人员、工具等)进行监测,选择传统分离培养方法对卫生指示菌和主要食源性致病菌进行检验,并对沙门菌进行血清学鉴定。结果原辅料中菌落总数10~5 CFU/g和大肠菌群10~3 CFU/g的样品比例分别为33.00%(33/100)和29.00%(29/100);中间产品中菌落总数10~5 CFU/g和大肠菌群10~3 CFU/g的样品比例分别为62.86%(66/105)和36.19%(38/105);终产品未检出菌落总数10~4 CFU/g的样品,大肠菌群均10 CFU/g。结论火腿肠加工过程存在微生物污染风险,本研究对掌握火腿肠加工过程的污染分布,确定关键控制点,为制定相关生产质量管理规范、确保终产品的食品安全具有重要意义。     

The efficacy of grape seed extract,citric acid and lactic acid on the inactivation of Vibrio parahaemolyticus in shucked oysters

The efficacy of grape seed extract (GE), citric acid (CA) or lactic acid (LA) on the inactivation of Vibrio parahaemolyticus in shucked oysters was studied. The minimum inhibitory concentration (MIC) of GE, CA or LA against V. parahaemolyticus in TSB-1% NaCl was also determined. The shucked oysters were artificially inoculated with V. parahaemolyticus, the inoculated shucked oysters (25 g) were then dipped in solution of GE (0.0, 10.0, 20.0, 50.0, 100, 200, 300 and 500 mg mL⁻¹), CA (0.0, 5.0, 10.0, 15.0, 20.0, 50.0, 100, 200 and 300 mg mL⁻¹) or LA (0.0, 1.0, 5.0, 10.0, 15.0, 20.0, 50.0, 100 and 150 mg mL⁻¹) for 10 min. The population of V. parahaemolyticus in shucked oysters was determined. The MICs of GE, CA or LA against V. parahaemolyticus were 10.0, 5.0 or 1.0 mg mL⁻¹, respectively. A 500, 300 or 150 mg mL⁻¹ GE, CA or LA solutions were needed to reduce the population of V. parahaemolyticus to below the detection level (1.0 log g⁻¹) in shucked oysters.     

Molecular biological characteristics of Staphylococcus aureus isolated from food

A survey was conducted to detect the distributing situation of Staphylococcus aureus in 6 kinds of food and the molecular biological characteristics of the isolates, so as to provide theoretical basis for our food risk monitoring. A total of 205 samples of 6 kinds of food were collected and examined for S. aureus. The GB 4789-2010 (food hygienic inspection method of China), mini VIDAS auto analysis, KB method and pulsed-field gel electrophoresis (PFGE) method were used to detect S. aureus, enterotoxin, drug sensitivity and homology analysis, respectively. The positive rates of S. aureus in the 205 samples were 15.6 % (32 samples), and the positive rates in raw milk were highest (30.0 %), with raw meat and aquatic products next and third (25.0and 12.0 %), cooked meat and milk products (10.0 % and 7.5 %, respectively). No S. aureus was detected in ice creams. With shock culturing in 37 °C for 24 h, 14 strains (43.7 %) produced enterotoxin in the 32 strains of S. aureus. In the drug sensitive tests, in the 32 isolates, 87.5 % were resistant to penicillin; 84.4 % were resistant to ampicillin; 26 (81.3 %) were resistant to tetracycline; 20 (62.5 %) showed resistance to erythromycin. All of the 32 strains were sensitive to trimethoprim sulfamethoxazole, oxacillin, cefoxitin, gentamicin, and vancomycin. No methicillin-resistant S. aureus was isolated from our food samples. PFGE typing showed that S. aureus genotypes profile had significant difference between the strains, indicating diversity contamination of foodborne S. aureus.     

Detection of Roundup Ready soy in highly processed products by triplex nested PCR

Till now, there still has not an effective detection method for the highly processed GM (genetically modified) products. A novel method of the triplex nested PCR, was developed for the sensitive detection of several foreign genes (Lectin, CaMV 35S, CTP, CP4-EPSPS, NOS) in highly processed products. We detected seven representative highly processed products (soya lecithin, soya protein powder, chocolate beverage, infant rice cereal, crude soybean oil, soybean refine oil, soybean salad oil) by the triplex nested PCR. The first triplex PCR cannot detect the insert signals in the processed products, and the sensitivity is 0.5%. However the second triplex PCR, which can simultaneously detect RR soybean targets with a sensitivity of 0.005% in the triplex nested PCR. The result indicates the advanced level of the method for the GM products detection. It is a flexible assay to detect the RR soybean in highly processed products.     

A 12-month longitudinal study of Listeria monocytogenes contamination and persistence in pork retail markets in China

Listeria monocytogenes is a foodborne pathogen frequently isolated from raw pork meat. This study was designed to investigate the prevalence and molecular characteristics of L. monocytogenes in raw pork from open markets in China. The survey was conducted monthly over a 12-month period in Zigong, China. L. monocytogenes was isolated from 262 of 1641 samples collected (16.0%) including minced meat samples (131/608, 21.5%), pork pieces samples (111/857, 13.0%) and environmental swabs (20/176, 11.4%). The isolation rates in spring and winter were significantly higher than those in summer and autumn (X² = 68.85, P < 0.05). All isolates were subjected to serotyping, multi-locus sequence typing (MLST) and AscI pulsed-field gel electrophoresis (PFGE). The 262 isolates were subtyped into five serotypes: 1/2b (43.1%), 1/2c (35.5%), 1/2a (19.1%), 4b (1.1%), 3a (1.1%); 20 sequence types (STs) with four most frequent STs, being ST9 (35.9%), ST87 (19.8%), ST3 (16.0%) and ST8 (14.1%); and 39 pulsotypes (PTs) with PT4 (26.3%), PT30 (14.5%) and PT11 (12.6%) being most frequent. Two primary pulsotypes from pork pieces were previously isolated from clinical listeriosis cases in the local hospitals. The six markets from different districts differed in the level of contamination and strain types. Persistent contamination of L. monocytogenes was found in the markets especially in meat mincers, which were found to be one likely source of continuous cross contamination. These findings will help develop strategies to reduce L. monocytogenes contamination in open markets for better public health control and prevention of foodborne L. monocytogenes infections.     

印迹膜电喷雾电离质谱法快速分析尿液中沙丁胺醇

沙丁胺醇(SAL)可促进动物生长,常被非法添加于畜禽饲料中,并可通过食物链进入人体,危害人体健康。目前,沙丁胺醇的分析方法已有很多报道,但对于复杂生物样本中沙丁胺醇的快速定量分析依然存在挑战。为实现这一目标,本研究采用印迹膜电喷雾电离质谱法(MIM-ESI MS)对尿液中的沙丁胺醇进行直接定性和定量分析。结果表明,该方法具有较高的灵敏度,尿液样本的检出限和定量限分别为5 ng/L和10 ng/L,在0.01~10 000 μg/L浓度范围内具有较好的线性关系。该方法无需样品前处理,其分子印迹膜材料具有特异性富集的功能,可实现生物复杂样本中痕量目标物的快速检测。