Xihong Zhao

Development and application of a novel multiplex polymerase chain reaction (PCR) assay for rapid detection of various types of staphylococci strains

In this study, a novel multiplex- polymerase chain reaction (PCR) for rapid detection of various staphylococci strains, including methicillin-resistant Staphylococcus aureus (MRSA), methicillin-sensitive Staphylococcus aureus, (MSSA) methicillin-resistant coagulase-negative staphylococci (MRCNS), methicillin-sensitive coagulase-negative staphylococci (MSCNS) and non-staphylococci strains, had been developed and applied. Six primers were specially designed on three target genes, which were mecA, 16S Ribosomal ribonucleic acid (rRNA) and femA. The specific amplification generated 3 bands on agarose gel, with sizes 374 bp for mecA, 542 bp for 16S rRNA and 823 bp for femA, respectively. The PCR product showed highest levels of resolution of DNA when 250 �M of dNTP, primer concentration of mecA, 16S rRNA and femA reaching 1, 1 and 3 �M respectively. No false positive amplification was observed, indicating the high specificity of the established multiplex PCR assay. Application of this multiplex-PCR had been further performed on detection for 262 MRSA and MRCNS strains with primers pairs M1 with M2 and F1 and F2. According to the results, multiplex-PCR results showed expected products for either MRSA or MRCNS strains, demonstrating the multiplex-PCR assays established in this study to be useful and powerful methods for differentiation of MRSA, MSSA, MRCNS, MSCNS and non-staphylococci strains.

Current Perspectives on Viable but Non-culturable State in Foodborne Pathogens

The viable but non-culturable (VBNC) state, a unique state in which a number of bacteria respond to adverse circumstances, was first discovered in 1982. Unfortunately, it has been reported that many foodborne pathogens can be induced to enter the VBNC state by the limiting environmental conditions during food processing and preservation, such as extreme temperatures, drying, irradiation, pulsed electric field, and high pressure stress, as well as the addition of preservatives and disinfectants. After entering the VBNC state, foodborne pathogens will introduce a serious crisis to food safety and public health because they cannot be detected using conventional plate counting techniques. This review provides an overview of the various features of the VBNC state, including the biological characteristics, induction and resuscitation factors, formation and resuscitation mechanisms, detection methods, and relationship to food safety.

Expression and purification of gp41-gp36 fusion protein and application in serological screening assay of HIV-1 and HIV-2

Recombinant fusion protein gp41-gp36 recognized by specific antibodies against HIV-1 and HIV-2, had been expressed and purified for the development of a serological screening kit in this study. Serological screening method by enzyme-linked immunosorbent assay (ELISA) with the purified gp41-gp36 was carried out to test the sensitivity and specificity of the protein by serum samples and reference panels from National Institute for the Control of Pharmaceutical and Biological Products (NICPBP), with the sensitivity and specificity found to be 100 and 99.0%, respectively. As the application was concerned, 42 HIV-1 positive,5 HIV-2 positive, and 300 negative human sera had been used, and reliable results had been obtained in comparison with other reference human immunodeficiency virus (HIV) ELISA kits in panels.

Research advance in rapid detection of foodborne Staphylococcus aureus

ABSTRACT Staphylococcus aureus is a gram-positive, coccus-shaped facultative anaerobe and a member of the Staphylococcaceae family. In recent years, alimentary toxicosis caused by S. aureus is a very serious problem worldwide, which constitutes a great threat to public health. In this review, we tried to summarize the conventional methods and newly developed rapid detection techniques of S. aureus (traditional detection method, biochemical detection, immunology method, molecular biology, and biosensor method) for their principles, advantages, disadvantages, and applications. Furthermore, the future perspectives of S. aureus detection methods were forecasted at last.

Intraspecific protoplast fusion of Brettanomyces anomalus for improved production of an extracellular β-glucosidase.

Improvement of production of an extracellular β-glucosidase with high activity by Brettanomyces anomalus PSY-001 was performed by using recursive protoplast fusion in a genome-shuffling format. The initial population was generated by ultraviolet irradiation, ultrasonic mutagenesis and, then, subjected to recursive protoplast fusion. Mutant strains exhibiting significantly higher β-glucosidase activities in liquid media were isolated. The best mutant strain showed increased cell growth in a flask culture, as well as increased β-glucosidase production. A recombinant strain, F3-25, was obtained after three rounds of genome shuffling and its production of β-glucosidase activity reached 4790 U L−1, which was a nearly eightfold increase compared to the original strain B. anomalus PSY-001. The subculture experiments indicated that F3-25 was genetically stable.

Detection of Foodborne Pathogens by Surface Enhanced Raman Spectroscopy

Food safety has become an important public health issue in both developed and developing countries. However, as the foodborne illnesses caused by the pollution of foodborne pathogens occurred frequently, which seriously endangered the safety and health of human beings. More importantly, the traditional techniques, such as PCR and enzyme-linked immunosorbent assay, are accurate and effective, but their pretreatments are complex and time-consuming. Therefore, how to detect foodborne pathogens quickly and sensitively has become the key to control food safety. Because of its sensitivity, rapidity, and non-destructive damage to the sample, the surface enhanced Raman scattering (SERS) is considered to be a powerful testing technology that is widely used to different fields. This review aims to give a systematic and comprehensive understanding of SERS for rapid detection of pathogen bacteria. First, the related concepts of SERS are stated, such as its work principal, active substrate, and biochemical origins of the detection of bacteria by SERS. Then the latest progress and applications in food safety, from detection and characterization of targets in label-free method to label method, is summarized. The advantages and limitations of different SERS substrates and methods are discussed. Finally, there are still several hurdles for the further development of SERS techniques into real-world applications. This review comes up with the perspectives on the future trends of the SERS technique in the field of foodborne pathogens detection and some problems to be solved urgently. Therefore, the purpose is mainly to understand the detection of foodborne pathogens and to make further emphasis on the importance of SERS techniques.

Detection of KRAS mutations of colorectal cancer with peptide-nucleic-acid-mediated real-time PCR clamping

ABSTRACT Colorectal cancer (CRC) is the third most common cancer in the world and its disease-specific mortality is estimated to be approximately 33% in the developed world. KRAS mutations have been shown to predict response to anti-EGFR (epidermal growth factor receptor) targeted monoclonal antibody therapy. Therefore, KRAS mutation testing of metastatic CRC patients is mandatory in the clinical setting to aid in the choice of appropriate therapy. Currently, the most common strategy for KRAS mutation detection consists of conventional polymerase chain reaction (PCR) and direct sequencing. However, it is a time-consuming and complicated procedure, not suitable for routine clinical test. The objective of this study is to develop and evaluate a highly sensitive and rapid method using peptide nucleic acid (PNA) oligomers mediated real-time PCR clamping for detection of KRAS mutations. The PNA-mediated PCR clamping assay can real-time detect a mutation in a sample containing 1% of the mutant allele in a mixture of wild-type genomic DNA, which also enables the accurate and rapid detection of all KRAS codon 12 and 13 mutations in a single reaction. The total assay time is short as it requires only 1.5 hours after the samples preparation. Thus, the present method offers a potential alternative to be applied in clinical samples of CRC for detection of DNA carrying KRAS mutations.

Induction of viable but nonculturable Escherichia coli O157:H7 by low temperature and its resuscitation

Viable but non-culturable (VBNC) cells are alive bacteria cells, but lose their culturability in conventional culture media, usually escape detection by the plate count method and pose a serious threat to food safety and public health. Therefore, it is urgent to study the VBNC status, and to provide theoretical basis and scientific basis for food processing and safety control caused by pathogenic microorganisms. In this study, Escherichia coli O157:H7 was induced to the VBNC state at two different temperatures (-20°C and 4°C) and its resuscitation and morphological changes under different nutritional conditions were studied. The initial inoculum of 2.1 × 107 CFU/mL E. coli O157:H7 cells were induced into the VBNC state in normal saline, distilled water, LB broth at -20 °C after 176, 160, 80 days, respectively. The results showed that E. coli O157:H7 reserved at -20°C, and LB culture medium were easier to enter VBNC state than others conditions, the cells still had metabolic activity and the cell morphology changed from the typical rod shape to short rod and the cell size decreased. The resuscitate ways including the direct warming resuscitation, gradual warming resuscitation, adding chemical substance resuscitation, and adding nutrients resuscitation were studied. The results showed that the optimal conditions of 5% Tween 80 and 3% Tween 80 acculated the resuscitation of E. coli O157:H7 VBNC state cells induced by low temperature LB medium and low temperature saline. E. coli O157:H7 VBNC state failed from resuscitation when incubating in LB broth, respectively using direct warming and adding nutrients substance. This study provides new insights into induction and resuscitation of VBNC E. coli O157:H7 and offers an approach for investigating the formation mechanism of VBNC foodborne pathogens in food safety.

Study the Features of 57 Confirmed CRISPR Loci in 38 Strains of Staphylococcus aureus

Staphylococcus aureus is a foodborne pathogen that causes food contamination and food poisoning, which poses great harm to health, agriculture and other hosts. Clustered regularly interspaced short palindromic repeats (CRISPR) are a recently discovered bacterial immune system that resists foreign genes such as phage DNA. This system inhibits the transfer of specific movable genetic elements that match the CRISPR spacer sequences, thereby preventing the spread of drug-resistant genes between pathogens. In this study, 57 CRISPR loci were screened from 38 strains of S. aureus based on the CRISPR database, and bioinformatics tools were used to investigate the structural features and potential functions of S. aureus CRISPR loci. The results showed that most strains contained only one CRISPR locus, a few strains contained multiple loci with sparsely distributed sites. These loci mainly included highly conserved direct repeat sequences and highly variable spacer sequences, as well as polymorphic cas genes. In addition, the analysis of secondary structure of direct repeat RNA showed that all sites can form stable RNA secondary structure. The results of constructing phylogenetic tree based on spacer sequence showed that some strains contained a high degree of phylogenetic relationship, while the differences among other strains in evolutionary processes were quite obvious. Of the 57 CRISPR loci identified, only the cas gene was found near the 4 CRISPR loci.

Specific Detection of Viable E.coli O157:H7 Cells by Coupling Propidium Monoazide with Loop-Mediated Isothermal Amplification

Traditional molecular detection methods cannot distinguish Escherichia coli O157:H7 in viable or dead state. In this study, the loop-mediated is other mal amplification (LAMP) method combined with propidium monoazide (PMA) treatment was developed to selectively detect the viable Escherichia coli O157:H7, but not dead cells. Four primers, including outer primers and inner primers, were specially designed for recognizing six distinct sequences on the species-specific rfbE gene of Escherichia coli O157:H7 genome. PMA penetrated selectively through the compromised cell membranes and intercalated into DNA, amplification of DNA from dead cells was inhibited completely by 3.0 μg/mL PMA, whereas the DNA derived from viable cells was amplified remarkably within 1 h by PMA-LAMP. This study offers a novel molecular detection method to distinguish between the viable and dead cells.