Real-Time Nucleic Acid Sequence-Based Amplification Assay for Detection of Hepatitis A Virus

A nucleic acid sequence-based amplification (NASBA) assay in combination with a molecular beacon was developed for the real-time detection and quantification of hepatitis A virus (HAV). A 202-bp, highly conserved 5′ noncoding region of HAV was targeted. The sensitivity of the real-time NASBA assay was tested with 10-fold dilutions of viral RNA, and a detection limit of 1 PFU was obtained. The specificity of the assay was demonstrated by testing with other environmental pathogens and indicator microorganisms, with only HAV positively identified. When combined with immunomagnetic separation, the NASBA assay successfully detected as few as 10 PFU from seeded lake water samples. Due to its isothermal nature, its speed, and its similar sensitivity compared to the real-time RT-PCR assay, this newly reported real-time NASBA method will have broad applications for the rapid detection of HAV in contaminated food or water.     

Real-time nucleic acid sequence-based amplification assay for detection of hepatitis A virus

A nucleic acid sequence-based amplification (NASBA) assay in combination with a molecular beacon was developed for the real-time detection and quantification of hepatitis A virus (HAV). A 202-bp, highly conserved 5' noncoding region of HAV was targeted. The sensitivity of the real-time NASBA assay was tested with 10-fold dilutions of viral RNA, and a detection limit of 1 PFU was obtained. The specificity of the assay was demonstrated by testing with other environmental pathogens and indicator microorganisms, with only HAV positively identified. When combined with immunomagnetic separation, the NASBA assay successfully detected as few as 10 PFU from seeded lake water samples. Due to its isothermal nature, its speed, and its similar sensitivity compared to the real-time RT-PCR assay, this newly reported real-time NASBA method will have broad applications for the rapid detection of HAV in contaminated food or water.     

Development of monoclonal antibody-coated immunomagnetic beads for separation and detection of norovirus (genogroup II) in faecal extract samples

Aims:  The aim of this study is to develop an RT-PCR assay combined with immunomagnetic beads (IMS/RT-PCR) coating monoclonal antibody (Mab) for separation and detection of norovirus (genogroup II) in faecal samples. We furthermore compare its detection limits with IMS/RT-PCR using polyclonal antibody (Pab) and the TRIzol extraction method followed by RT-PCR (TRIzol-RT-PCR).
Methods and Results:  Mab-coated beads and Pab-coated beads were added to a series of tenfold dilutions of faecal extract containing norovirus in 1 ml PBS. After incubation and collection, the RNA was released by heating from virus separated by beads. The tenfold dilutions of faecal were also extracted with TRIzol reagent. The RNA was used as the template for RT-PCR detection (primers: JV12–JV13). IMS/RT-PCR using Mab showed an endpoint in the 10⁻⁷ dilution and was 10² times more sensitive than IMS/RT-PCR using Pab and was at least 10³ times more sensitive than TRIzol-RT-PCR method.
Conclusions:  IMS/RT-PCR using Mab proved to be a more sensitive method of noroviruses (NVs) detection than IMS/RT-PCR using Pab and the TRIzol-RT-PCR method.
Significance and Impact of the Study:  This is the first study to detect NVs with IMS/RT-PCR using Mab, and could serve as a model for future assays when broadly reactive NVs-specific Mabs are developed.     

Detection of replication-defective hepatitis A virus based on the correlation between real-time polymerase chain reaction and ELISA in situ results

ELISA in situ can be used to titrate hepatitis A virus (HAV) particles and real-time polymerase chain reaction (RT-PCR) has been shown to be a fast method to quantify the HAV genome. Precise quantification of viral concentration is necessary to distinguish between infectious and non-infectious particles. The purpose of this study was to compare cell culture and RT-PCR quantification results and determine whether HAV genome quantification can be correlated with infectivity. For this purpose, three stocks of undiluted, five-fold diluted and 10-fold diluted HAV were prepared to inoculate cells in a 96-well plate. Monolayers were then incubated for seven, 10 and 14 days and the correlation between the ELISA in situ and RT-PCR results was evaluated. At 10 days post-incubation, the highest viral load was observed in all stocks of HAV via RT-PCR (10⁵ copies/mL) (p = 0.0002), while ELISA revealed the highest quantity of particles after 14 days (optical density = 0.24, p < 0.001). At seven days post-infection, there was a significant statistical correlation between the results of the two methods, indicating equivalents titres of particles and HAV genome during this period of infection. The results reported here indicate that the duration of growth of HAV in cell culture must be taken into account to correlate genome quantification with infectivity.     

Time-resolved fluorescence immunoassay (TRFIA) for the detection of Escherichia coli O157:H7 in apple cider.

An immunoassay based on immunomagnetic separation and time-resolved fluorometry was developed for the detection of E. coli O157:H7 in apple cider. The time-resolved fluorescent immunoassay (TRFIA) uses a polyclonal antibody bound to immunomagnetic beads as the capture antibody and the same antibody labeled with europium as the detection antibody. Cell suspensions of 10(1) to 10(8) E. coli O157:H7 and K-12 organisms per ml were used to test the sensitivity and specificity of the assay. The sensitivity of the assay was 10(3) E. coli O157:H7 cells with no cross-reaction with K-12. Pure cultures of E. coli O157:H7 (10(1) to 10(5) CFU/ml) in apple cider could be detected within 6 h, including 4 h for incubation in modified EC broth with novobiocin and 2 h for the immunoassay. When apple cider was spiked with 1 to 10(3) CFU/ml of E. coli O157:H7 and 10(6) CFU/ml of K-12, our data show that the high level of K-12 in apple cider did not impede the detection of low levels of O157:H7. The minimum detectable numbers of cells present in the initial inoculum were 10(2) and 10(1) CFU/ml after 4- and 6-h enrichment. The TRFIA provides a rapid and sensitive means of detecting E. coli O157:H7 in apple cider.     

Capturing B type acute lymphoblastic leukemia cells using two types of antibodies

One way to monitor minimal residual disease (MRD) is to screen cells for multiple surface markers using flow cytometry. In order to develop an alternative microfluidic based method, isolation of B type acute lymphoblastic cells using two types of antibodies should be investigated. The immunomagnetic beads coated with various antibodies are used to capture the B type acute lymphoblastic cells. Single beads, two types of beads and surface immobilized antibody were used to measure the capture efficiency. Both micro and nanosize immunomagnetic beads can be used to capture B type acute lymphoblastic cells with a minimum efficiency of 94% and maximum efficiency of 98%. Development of a microfluidic based biochip incorporating immunomagnetic beads and surface immobilized antibodies for monitoring MRD can be an alternative to current cost and time inefficient laboratory methods. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2737, 2019     

检测大肠杆菌(Escherichia coli) O157:H7免疫磁珠的制备、保存与应用

【背景】大肠杆菌(Escherichia coli) O157:H7是导致肠出血性大肠杆菌食源性疾病暴发的主要血清型,免疫磁珠(Immunomagnetic beads,IMBS)在E. coli O157的检测中发挥着重要作用,而免疫磁珠的稳定性、特异性、广谱性等性能指标关系着在实际应用中的使用效果。【目的】制备高效、稳定且具有广谱性的免疫磁珠,联合分子检测技术如环介导恒温扩增 (Loop-mediated isothermal amplification,LAMP)技术、PCR等,提高目标菌的检出率。【方法】采用新型的磁珠活化剂MIX&GO制备E. coli O157免疫磁珠,并进行广谱性以及特异性检测;针对6种试剂牛血清白蛋白(Bovine serum albumin,BSA)、酪蛋白(Casein)、海藻糖(Trehalose)、聚乙烯吡咯烷酮(Polyvinyl pyrrolidone,PVP)、抗坏血酸(Vitamin C)和防腐剂ProClin 300,利用正交试验L18(37)优化免疫磁珠保存液组分;采用IMBS-LAMP、IMBS-PCR、IMBS-生化、菌液-LAMP、菌液-PCR、显色平板-生化鉴定6种方式对20份生猪肉样品进行检测。【结果】利用MIX&GO活化剂制备的免疫磁珠捕获率最高达到81.5%±1.3%;免疫磁珠保存液最优配方为：牛血清白蛋白15.0 g/L,酪蛋白10.0 g/L,海藻糖10.0 g/L,PVP 2.0 g/L,抗坏血酸5.0 g/L,ProClin 300 2.5 g/L,保存6个月后免疫磁珠捕获率为75.5%;在20份生猪肉样品的检测中,自制磁珠和商品化磁珠与LAMP联用均检出9例阳性样品;IMBS-LAMP在6种检测方式中具有最高的检测灵敏度,但检出的样品会因磁珠抗体的差异而有所不同。【结论】与商品化磁珠相比,实验制备的免疫磁珠具有良好的特异性和广谱性,免疫磁珠-LAMP联用提高了目标菌的检出率,是一种高灵敏度、具有应用前景的检测方法。     

Capture antibody targeted fluorescence in situ hybridization (CAT-FISH): dual labeling allows for increased specificity in complex samples

Pathogen detection using biosensors is commonly limited due to the need for sensitivity and specificity in detecting targets within mixed populations. These issues were addressed through development of a dual labeling method that allows for both liquid-phase fluorescence in situ hybridization (FISH) and capture antibody targeted detection (CAT-FISH). CAT-FISH was developed using Escherichia coli O157:H7 and Staphylococcus aureus as representative bacteria, and processing techniques were evaluated with regard to FISH intensities and antibody recognition. The alternative fixative solution, methacarn, proved to be superior to standard solid-phase paraformaldehyde fixation procedures, allowing both FISH labeling and antibody recognition. CAT-FISH treated cells were successfully labeled with FISH probes, captured by immunomagnetic separation using fluorescent cytometric array beads, and detected using a cytometric array biosensor. CAT-FISH treated cells were detectable with LODs comparable to the standard antibody-based technique, (~ 10³ cells/ml in PBS), and the technique was also successfully applied to two complex matrices. Although immunomagnetic capture and detection using cytometric arrays were demonstrated, CAT-FISH is readily applicable to any antibody-based fluorescence detection platform, and further optimization for sensitivity is possible via inclusion of fluorescently tagged antibodies. Since the confidence level needed for positive identification of a detected target is often paramount, CAT-FISH was developed to allow two separate levels of specificity, namely nucleic acid and protein signatures. With proper selection of FISH probes and capture antibodies, CAT-FISH may be used to provide rapid detection of target pathogens from within complex matrices with high levels of confidence.     

DETECTION OF LISTERIA IN FOOD AND ENVIRONMENTAL SAMPLES BY IMMUNOMAGNETIC BEAD CAPTURE AND BY CULTURAL METHODS

Detection of Listeria by capture on antibody-coated magnetic beads has been shown to decrease test time and improve sensitivity, relative to cultural methods, in a study of spiked environmental samples (Mitchell et al. 1993). In this study, immunomagnetic capture was compared to standard cultural methods for detection of Listeria in a broad range of spiked and naturally contaminated food and environmental samples. Immunomagnetic capture was at least as sensitive as cultural methods for detection of Listeria in seafood, meats, dairy foods, and environmental samples. It was possible to determine the number of Listeria present in a sample, because immunomagnetic capture was carried out directly from the sample, without enrichment. These quantitative results were produced within 24 h, while cultural methods required 6–14 days to produce a qualitative result. Immunomagnetic capture was thus more rapid and as sensitive as standard cultural methods for detection of Listeria in the food and environmental samples tested.     

Duration of viral RNA circulation in blood of patients with hepatitis A

Duration of hepatitis A virus (HAV) RNA circulation in blood of patients with HA was assessed and compared with intensity of cytolytic syndrome. Detection of viral RNA was performed by RT-PCR method with specific primers to VP1/P2A region of HAV genome. 54 blood serum samples from 40 patients were prospectively studied on the presence of HAV RNA. The latterwas detected in 53.7% of serum samples. The greatest number of positive results of HAV RNA detection in blood of the patients with HA was obtained from 8th to 21st day of illness (77.4%). Prolonged viremia (42+/-9 days) was observed in more than 20% of the patients. The maximal time of HAV RNA daetection in blood serum amounted 74 days (period of follow-up). HAV RNA was present in almost all patients with AIAT activity higher than 500 U/l regardless of duration of illness.     

基于免疫磁分离的三重荧光定量PCR检测食品中沙门氏菌、志贺氏菌和金黄色葡萄球菌

【目的】开发一种同时对食品中沙门氏菌、志贺氏菌和金黄色葡萄球菌快速、灵敏、准确的检测方法。【方法】利用特异性免疫磁球,在37°C条件下从250 m L猪肉增菌液体系中边富集边循环捕获目标菌。快速提取DNA后,利用特异性的引物与探针,对3种食源性致病菌进行三重荧光定量PCR检测。【结果】针对沙门氏菌、志贺氏菌和金黄色葡萄球菌的检测限分别达到2.0、6.8和9.6 CFU/g。方法总体灵敏度、特异性和准确度达到99.2%、100%及99.5%。对151份实际样品进行检测,与国标(GB/T 4789.4-2010、GB 4789.5-2012和GB/T4789.10-2010)方法的检测结果相比,金黄色葡萄球菌有一例阴性偏差。【结论】开发的基于免疫磁分离的三重荧光定量PCR方法,能够在8 h内完成对食品中3种致病菌检测,并且灵敏度高、特异性好、检测准确,可以作为快速应对此类食品安全突发事件的检测手段。