Current Biotechnology

Research Progress on the Flow Cytometry Counting for Single Nucleotide Molecule
WANG Di, WU Xiao, WANG Zhidong, GAO Yunhua^*
2020, 10(6): 573-578. DOI: 10.19586/j.2095-2341.2020.0101
Abstract ( ) PDF (1503KB)( )

Developing accurate and sensitive nucleotide quantification method to improve the detection capability for traceable nucleotide samples, is of great importance under the background of fighting against present COVID-19 epidemic. Flow cytometry counting for single nucleotide molecule is a type of numeration-based quantification method, which counts the copy number of target fluorescence labelled-nucleotide molecules without PCR amplification, tracing directly to international system of unit. As a potential primary metrological method, it is an important supplement for the present nucleotide quantification method, and could be widely applied for many fields, such as clinical diagnostics, genetic-modified organism labelling and detection of food & drug residues. Here the principal, hardware constitution of the apparatus, data analysis model were reviewed, and the significance to biometric standard system was also prospected.

Application of Digital PCR Technology in the Development of Nucleic Acid Reference Materials
ZHENG Zifan, LIU Fangfang, LIU Weixiao, JIN Wujun, LI Liang^*
2020, 10(6): 579-584. DOI: 10.19586/j.2095-2341.2020.0128
Abstract ( ) PDF (1449KB)( )

In the field of reference material development, the development of biological reference material has gradually become a hot topic. At the same time, the development and application of nucleic acid-based detection technology has promoted the process of nucleic acid reference material. Nucleic acid reference materials require high-level and accurate valuation methods, and digital PCR has been widely used as a single-molecule quantitative technology. Digital PCR is an absolute quantitative method for the determination of nucleic acid molecules. For example, droplet digital PCR uses droplets formed by water-in-oil as the reaction chamber, and distributes the reaction solution containing the DNA template to a large number of independent reaction chambers for expansion. Increase the reaction, and then quantify the copy number of DNA by counting the positive signals in the reaction chamber, so as to achieve the purpose of accurately quantifying the copy number of nucleic acid. This article reviewed the literature on the research of digital PCR and the latest application progress of digital PCR technology in the development of DNA and nucleic acid reference materials, especially in the fields of medical diagnosis and genetic modification detection, which was expected to provide reference for the research of reference material development.

Research on Calibration Method of Digital PCR Instrument
WANG Shangjun¹, DONG Lianhua²
2020, 10(6): 585-589. DOI: 10.19586/j.2095-2341.2020.0106
Abstract ( ) PDF (2053KB)( )

The digital PCR instrument is an important instrument for absolute quantification of nucleic acids, and it is of great significance to ensure the accuracy of the detection results of the digital PCR instrument. Different digital PCR instruments on the domestic market were compared, the performance indexes of the digital PCR instrument were analyzed and the calibration method of the instrument were discussed. Several key measurement technical indicators of the digital PCR machine calibration were set, including the copy number concentration relative indication error, copy number concentration repeatability, fluorescence channel consistency and the number of reaction unit repeatability. The method was tested and verified by utilizing traceable national certified reference materials. The verification results showed the feasibility of the calibration method. The method has strong operability, can meet the technical requirements of the instrument and the needs of users, improve the accuracy and reliability of the detection results of the digital PCR instrument, and has a positive role in further expanding and deepening the application of the digital PCR technology.

Research Progress of Reference Material for HPV Nucleic Acid Detection
YANG Jiayi1§*, CHEN Guifang2§, GAO Yunhuan1, WANG Zhidong1, WU Xiao1
2020, 10(6): 590-596. DOI: 10.19586/j.2095-2341.2020.0098
Abstract ( ) PDF (1115KB)( )

According to the global cancer statistics in 2018, the incidence of cervical cancer in China has ranked the second in the world, and it tends to occur at a younger age, posing a serious threat to women health. Research has shown that the infection of high-risk human papillomavirus (HPV) closely relates to the occurrence of cervical cancer. Hence, the detection of HPV is of great significance for the prevention and diagnosis of cervical cancer. The popular detection methods work based on PCR technology, which make qualitative and quantitative analysis of HPV nucleic acid. However, in the absence of uniform standards, it is difficult to ensure accurate and comparable measurement results. Reference material acts as a powerful tool for measurement and calibration, which could be applied to quality control of measurement process. This paper discussed the common detection methods for HPV and the influencing factors in the detection procedure, then analyzed the characteristics of different forms of reference materials for nucleic acid detection. It was expected to provide clues for the research and usage of the reference materials in HPV nucleic acid detection, which would be helpful for the scientific diagnosis of cervical cancer.

Review on Research Progress of Protein Content Metrology Technology
WANG Xianxia1,2, WU Liqing2*, YANG Bin2, ZHANG Ning2, SU Ping1*, YANG Yi1
2020, 10(6): 597-606. DOI: 10.19586/j.2095-2341.2020.0139
Abstract ( ) PDF (1896KB)( )

The 21st century is the century of life science. With the progress of life science and the development of biological industry, the demand for accurate and comparable measurement results has given birth to the emergence and development of protein metrology. After development of nearly 20 years, protein content metrology technology has made a breakthrough and made considerable progress. In this paper, the research progress and breakthroughs in protein content metrology technology were reviewed. The applications of mass balance, isotope dilution-mass spectrometry, quantitative nuclear magnetic resonance, electrospray-differential electromigration-particle counting, high performance liquid chromatography-circular dichorism in protein content metrology were introduced. Finally, the future development of protein content metrology technology was prospected.

Review on Research Progress of Protein Activity Metrology Technology
WANG Xianxia1,2, WU Liqing2*, YANG Bin2, ZHANG Ning2, SU Ping1*, YANG Yi1
2020, 10(6): 607-612. DOI: 10.19586/j.2095-2341.2020.0151
Abstract ( ) PDF (2518KB)( )

With the development of science and the progress of society, life science has developed from the description of phenomena to the stage of precise quantitation. The international protein metrology technology has also made considerable progress. Nowadays, a framework for protein metrology has been initially constructed, and the corresponding value transfer method has been established, forming a basically fixed research model. This article reviewed the research progress and breakthroughs of protein activity metrology technology so far, focusing on metrology technology and application of catalytic activity concentration and immunoaffinity concentration. Finally, the future development direction of protein activity metrology technology was summarized and prospected.

Progress on Development and Detection of Internal Reference Genes in Six Crops
GAO Jiaqi1, CHEN Shuo1, WANG Di2, LONG Yan2, LI Liang2*, ZHANG Xiao1*
2020, 10(6): 613-622. DOI: 10.19586/j.2095-2341.2020.0127
Abstract ( ) PDF (1176KB)( )

Internal reference gene refers to a conserved DNA sequence which can distinguish species specificity, has a single or constant low copy number, and low variation. The internal reference genes of designated crop species are used as the main genetic markers to identify other species in many fields, such as transgenic component detection, species and product discrimination, etc. Especially in the quantitative and qualitative detection of genetically modified products, they can be used to detect species sources and transgenic content in samples, which is of great significance to the quantitative and qualitative detection of genetically modified products. At present, many kinds of internal reference genes of crops have been developed, among which the number of internal reference genes developed for maize, rape and rice is relatively large, but there is still a lack of more thorough comparative research, which makes it difficult to select and apply them. Based on this, according to the existing research results at home and abroad, the research progress of internal reference genes of six crops (including corn, soybean, rape, cotton, rice and wheat) with great demand for detection and identification was summarized, and the internal reference genes of common crops were systematically compared and studied, in order to provide technical support for plant-derived and transgenic components and related detection and identification.

Progress on Reference Materials Based on Mass Balance Method
ZHENG Zifan, LIU Weixiao, JIN Wujun, LI Liang^*
2020, 10(6): 623-629. DOI: 10.19586/j.2095-2341.2020.0113
Abstract ( ) PDF (1146KB)( )

In order to ensure the comparability of measurement results in different regions and at different times, the measurement results need to be traced to appropriate and prescribed reference standards. For the measurement of materials and samples in the fields of chemistry, biology, engineering and physics, the reference standard is a standard substance. It can be seen that the determination of reference materials is very important for the detection and quantification of materials. Reference material (RM) is a sufficiently uniform material or substance with one or more characteristic values that are relatively easy to determine. It can be used to assign values to materials, evaluate measurement methods, and calibrate measurement instruments. As one of the quantitative methods of reference materials, mass balance method is a commonly used purity measurement method, which deducts the content of impurities such as moisture, ash, volatile components and inorganic elements from 100%, and then determines the purity of materials according to the percentage of main components in organic components. The mass balance method has high accuracy and can be traced to the quality units in the international system of units. If the main components and various impurities in the sample are measured by the benchmark method to complete the measurement of the whole mass balance method, the mass balance method is expected to become a new benchmark method. Based on this, the principle of mass balance method and its application in the development of reference materials were introduced, and the latest application of mass balance method in reference materials was summarized in order to explore more possibilities of mass balance method in the development of reference materials.

Preparation of Plasmid DNA Reference Material Containing Human STR D6S1043-1 Fragment
YAN Anxin1, CHEN Jing1, LIU Hongdi1, LI Yongjiu1, PENG Zhu1, ZHAO Hemiao1, LI Liang2, ZHAO Xingchun1*
2020, 10(6): 630-636. DOI: 10.19586/j.2095-2341.2020.0146
Abstract ( ) PDF (3276KB)( )

Short tandem repeat (STR) is a kind of DNA sequence with length polymorphism existing in human genome, which consists of 2~6 base pairs of repeating units connected in series. DNA STR typing test is the main basis for individual identification and genetic identification in forensic science at present. STR typing reference materials are the basis and key materials for the traceability of DNA STR test, and the research on them will greatly promote the standardization process of forensic DNA STR test in China. Taking STR locus D6S1043 as an example, the preparation process of plasmid DNA reference materials for STR sequences from human genome was introduced. At first, a DNA sequence (defined as D6S1043-1) containing 13 repeat units (\[ATCT\]13) of the D6S1043 locus was synthesized and ligated to pUC57 vector to construct a recombinant plasmid, thus preparing a plasmid solution. Secondly, the accuracy of DNA sequence was tested by enzyme digestion identification, Sanger sequencing, and typing identification of multiple STR typing kits. Thirdly, the concentration of plasmid was detected by using droplet digital PCR (ddPCR) method, as well as the uniformity and stability of plasmid solution. Finally, the concentration standard value was determined through the cooperation of 8 laboratories, and the total uncertainty was obtained through comprehensively calculating the three kinds of uncertainties introduced by the homogeneity test, stability test and the determination process. And the typing value of D6S1043-1 was confirmed by typing identification of multiple STR typing kits in 6 laboratories. The results showed that the reference material had good uniformity and stability, and could be stored for 6 months at -20 ℃ and 14 d at 4 ℃; the concentration of the plasmid was (7.7±1.2)×103 copies·μL-1; the typing value was 13 at the D6S1043 locus. Human D6S1043-1 STR plasmid DNA reference material \[code: GBW(E)091072\] had become one of the first certified reference materials in the field of forensic DNA test, whose preparation process provided a certain reference for the preparation of plasmid DNA reference materials for other STR loci.

Study on Authenticity Identification Method of Honeysuckle Based on Pyrosequencing Analysis Technology
XU Shiyong1, ZHAO Xin1, ZHANG Fuli2, LIU Zhenghui1, SHI Qinghong1, SONG Jun3, WANG Yong1, LAN Qingkuo1*
2020, 10(6): 637-645. DOI: 10.19586/j.2095-2341.2020.0114
Abstract ( ) PDF (7997KB)( )

Honeysuckle is a staple medicine, adulteration in the market occurs sometimes, but the present detection method could not meet the requirements, therefore, it is urgent to establish a fast and accurate detection method. Combined with SNP labeling and pyrosequencing typing analysis technology, PCR amplification and pyrosequencing analysis were performed on the different SNP sites of Flos lonicerae and its common counterfeits, and an accurate and high-throughput method was established to detect the adulteration amount of Flos lonicerae. Results showed that, the signature SNP sites of Flos lonicerae and its counterfeits were quantitatively analyzed by pyrosequencing technology, and the authenticity and adulteration amount of Flos lonicerae were identified. This method has the sensitivity of PCR technology and the accuracy of sequencing technology, and is characterized by good repeatability, high degree of automation and simple operation. It can complete the detection of the adulteration amount of honeysuckle components within three hours and obtain the quantitative results.

Application Progress of Next Generation and the Third Generation Sequencing Technology in Genetic Diagnosis
GAO Haiming, ZHAO Yanyan^*
2020, 10(6): 646-654. DOI: 10.19586/j.2095-2341.2020.0100
Abstract ( ) PDF (1189KB)( )

Prevention and treatment of genetic diseases is a major issue in public health, and it is an important part to clarify the genetic causes. High-throughput sequencing technology (also known as second generation sequencing technology) has the advantages of high throughput, low cost and high accuracy, which provides direct evidence for genetic diagnosis and consultation, and has become an indispensable powerful tool for genetic testing. The third generation sequencing also occupies a place in clinical application by virtue of its unique advantages of long reading time. Next generation and the third generation sequencing technologies have their own characteristics and complement each other. There are many types of sequencing schemes to choose from according to different detection requirements in clinic. Based on this, the principle, classification and application progress of the second and third generation sequencing technologies in genetic diagnosis were reviewed in order to provide ideas and guidance for the selection of clinical sequencing schemes.

Application and Prospective of Nanomaterials in Crop Research
XUE Qinqin1,2, HAN Beibei1, WU Xueqing1, LI Pei1, LI Yingying1,3, WU Qingyu1*
2020, 10(6): 655-660. DOI: 10.19586/j.2095-2341.2020.0118
Abstract ( ) PDF (1095KB)( )

In recent years, nano-materials have become a research hotspot in the field of agriculture, and have been gained much attention internationally. Nanomaterials, because of their small size, can be absorbed by cells through endocytosis after penetrating the cell walls, and then affect the growth of plants. Nanomaterials are widely used in the crop research fields such as plant genetic transformation, crop growth and health, especially in the field of genetic transformation, which can be used as a transformation vector and integrated with gene editing technology for genetic improvement of crops. Based on this, the absorption and transport mechanism of nanomaterials in plants and their application in crops were reviewed, with emphasis on the research progress of nanomaterials in plant genetic transformation. The remaining issues and the future development direction of nanomaterials in crops were prospected, in order to provide reference for broadening the application of nanomaterials.

DNA Tetrahedral Nanostructure and its Application Progress in Biotechnology
CHEN Shuo1, GAO Jiaqi1, WANG Di2, LONG Yan2, LI Liang2*, ZHANG Xiao1*
2020, 10(6): 661-667. DOI: 10.19586/j.2095-2341.2020.0122
Abstract ( ) PDF (2163KB)( )

DNA tetrahedral nanostructure is a kind of DNA three-dimensional nanostructure with tetrahedral shape, which is formed by precise and ingenious DNA sequence design, applying the principle of base complementary pairing and automatic hybridization of four single strands. It has good biocompatibility, excellent cell membrane permeability, simple preparation, high yield, adjustable size and dynamics, so it has been widely studied and applied in the fields of microbial identification, medical diagnosis and biosensors. Based on this, DNA tetrahedral nanostructures and their functional modification were introduced, and the application progress of DNA tetrahedral nanostructures in biotechnology was summarized, in order to provide reference for promoting the research of DNA tetrahedral nanostructures and broadening their application fields.

Pyrosequencing-based Detection Method of Gene Edited Rice
XIE Meixia1,2, WANG Yan1, ZHAO Xin1, GAO Yue1, LIU Shuang3, CHEN Rui1, LAN Qingkuo1, XU Xiaohui4, CAO Jijuan5, WANG Yong1*
2020, 10(6): 668-673. DOI: 10.19586/j.2095-2341.2020.0123
Abstract ( ) PDF (3505KB)( )

Gene editing technology is developing rapidly, but there are few related detection methods. In order to find a detection method for gene-edited crops, this study used editing sites of PL3 gene-edited rice as the target, effectively designed pyrosequencing amplification primers and sequencing primers, and conducted effectiveness testing, respectively using Sequence to Analyze and other programs as well as the two models of SNP and AQ, the qualitative and quantitative detection test of PL3 gene has been completed, and the pyrosequencing detection method on editing site of PL3 gene-edited rice has been established. The results showed that the editing type rice could be distinguished from the wild type rice by detecting the editing sites based on pyrosequencing. Compared with the conventional methods, the method has the advantages of better accuracy, high efficiency and high sensitivity. It has a good application prospect in qualitative and quantitative detection of editing sites at the gene editing rice.

Application Prospects of Digital PCR in Detection of SARS-CoV-2
HU Sihong, YOU Guoye
2020, 10(6): 674-679. DOI: 10.19586/j.2095-2341.2020.0070
Abstract ( ) PDF (1322KB)( )

In 2020, corona virus disease 2019 (COVID-19) pandemic caused by SARS-CoV-2 outbreaks in many countries around the world, which poses a great challenge to the prevention and control of the epidemic. Nucleic acid detection by real-time fluorescent PCR(RT-PCR) is the main detection method used at this stage, but the situation of “false negative results”, multiple sampling and repeated detection is also frequent. There is an urgent need to establish a detection technology with higher sensitivity, less sample need and simple operation to screen out patients infected SARS-CoV-2. Digital PCR has shown promise as a precision nucleic acid quantification technology because of its high sensitivity and specificity, which has been widely used in the field of virus detection. In this paper, the structure of SARS-CoV-2 and the problems of detecting methods by RT-PCR were introduced. And the application prospects of digital PCR in the detection of the SARS-CoV-2 were described, which was expected to provide references for the development of more efficient detection kits.

Establishment and Application of Nucleic Acid Chromatography for Rapid Detection of Transgenic Plants
REN Wen1, YANG Haixia2, CHEN Lizhu2, LI Yufeng2, LIU Ya1*
2020, 10(6): 680-687. DOI: 10.19586/j.2095-2341.2020.0105
Abstract ( ) PDF (7775KB)( )

With the acceleration of transgenic research and industrialization in China, the importance of transgenic detection has become increasingly prominent. Therefore, the research and development of new methods for rapid and efficient detection has great significance in production and scientific research. In transgenic detection, conventional PCR technology has the characteristics of wide detection range, but it is time-consuming and laborious, and has high requirements for experimental conditions. Meanwhile, colloidal gold protein test paper method is convenient and fast, but the detectable range is narrow. Based on this, a set of method system for rapid transgenic detection based on nucleic acid chromatography was established. After extracting DNA from the samples by liquid ammonia by one-tube method, PCR amplification was carried out directly, and then the PCR amplification products were dripped onto the colloidal gold detection card, and the test results were observed by eyes directly due to the clear difference of color. In this method, corn internal reference genes ZSSⅡB and Zein, soybean internal reference gene SPS, rice internal reference gene Lectin, transgenic element promoter CaMV35S and terminator NOS, insect-resistant genes Cry1Ab/1Ac, herbicide-resistant genes Bar, Pat, CP4-EPSPS and selectable marker genes NPTⅡ and Pmi were finally detected. And the specific transgenic events Mir604 and Bt11 were also successfully detected. The nucleic acid chromatography detection method obtained by the research had the advantages of high sensitivity, efficient, and low requirements for detection conditions. It combined the advantages of PCR and protein test paper detection, and could be widely used in accurate and rapid detection of genetically modified products, providing a good technical support for the safety supervision of genetically modified organisms in China.

Qualitative PCR Methods for the Detection of Transgenic Herbicide-tolerant Maize G1105E-823C
WEN Hongtao1§,YANG Yang1§,DING Yijia1,YUAN Ran1,ZHANG Xiujie2,ZHANG Ruiying1*
2020, 10(6): 688-695. DOI: 10.19586/j.2095-2341.2020.0138
Abstract ( ) PDF (6699KB)( )

Transgenic herbicide-resistant maize G1105E-823C is a new glyphosate-resistant transgenic maize line with modified mG2-aroA gene. It has a higher tolerance to glyphosate-herbicide. At present, this strain has completed production test and has important industrial application prospect in China. However, there is no relevant report on the specific detection method of transformants for G1105E-823C, which is very unfavorable for the detection and supervision of this strain. Based on this, the ordinary PCR and real-time fluorescence PCR qualitative detection methods of transgenic herbicide-resistant maize G1105E-823C were established with the specific sequence of G1105E-823C transformant as the target. The results showed that both methods could detect the components of transgenic herbicide-resistant maize G1105E-823C transformants with high specificity. The detection limit of ordinary PCR detection method was 0.1%, and that of real-time fluorescence PCR detection method was 0.05%. The two qualitative detection methods established in this study provided new technical means for the accurate detection of transgenic herbicide-resistant maize G1105E-823C, and provided technical support for agricultural transgenic supervision.

Establishment of Transformant-specific Detection Method for the Herbicide-tolerant Transgenic Soybean Event ZUTS-33 Harboring the g10-epsps Transgene
MIAO Qingmei1, ZHAO Yang2, XU Xiaoli1, XU Junfeng1, WANG Xiaofu1, CHEN Xiaoyun1*
2020, 10(6): 696-703. DOI: 10.19586/j.2095-2341.2020.0124
Abstract ( ) PDF (3814KB)( )

ZUTS-33 is the transgenic soybean line developed by Zhejiang University with the modified agronomic trait of herbicide tolerance, which has entered the stage of safety assessment before commercial release. So far, there is no literature report on the detection method of this new transgenic variety, so it is urgent to establish an accurate quantitative detection method to provide technical support for the safety management of agricultural genetically modified organisms. Primers and TaqMan probes were designed according to the specific sequences of the transgene and the insertion site of the T-DNA in herbicide-resistant soybean ZUTS-33 genome. The specificity, accuracy, precision and repeatability of the primer pairs and probes were evaluated by the optimized real-time fluorescence quantitative PCR detection method, and the detection limit of detection (LOD) and quantitative limit (LOQ) of this detection method were determined. The experimental results showed that the established real-time fluorescence quantitative PCR detection method for transformant specificity of transgenic soybean ZUTS-33 had high specificity of line identification, and accuracy and precision met the requirements, and repeatability was good. LOD and LOQ of the detection method reached 20 copies and 40 copies respectively. The results provided an effective method for identification, detection and monitoring of transgenic soybean ZUTS-33 with the g10-epsps gene.

Loop-mediated Isothermal Amplification Method for Detection of Bovine-related Species Based on the Specific DNA Sequence
QU Zhan, JIA Ben, YANG Litao^*
2020, 10(6): 704-710. DOI: 10.19586/j.2095-2341.2020.0069
Abstract ( ) PDF (7982KB)( )

In recent years, adulteration of meat products frequently occurs, it is urgent to establish a rapid and reliable detection method for animal-derived components in meat products to ensure the safety of meat products. Compared with conventional PCR and other detection methods, loop-mediated isothermal amplification (LAMP) method has the advantages of high specificity, high sensitivity, quick reaction and easy operation. Based on this, the whole genome sequences of common cattle and their related species (yak, buffalo, bison) were screened by bioinformatics algorithm to obtain the similarity sequences among species, and then the specific LAMP method for detecting the components of common cattle and their related species was established and optimized. The final optimized LAMP reaction system was as follows: 0.25 μL of 10 μmol·L-1 outer primers F3 and B3, 2 μL of 10 μmol·L-1 inner primers FIP and BIP, 2.5 μL of 2 mmol·L-1 dNTP, 4 μL of 25 mmol·L-1 MgSO4, 3.5 μL of 5 mol·L-1 betaine, 1 μL of 8.0 U·μL-1 Bst DNA polymerase, 2.5 μL of 10×ThermoPol Buffer, 2 μL of DNA template, and adding double distilled water to 25 μL. The reaction conditions of LAMP were: amplification at 65 ℃ for 1 h and then amplification at 80 ℃ for 10 min. After adding SYBR GreenⅠfluorescent dye into the amplification product, the detection results could be directly observed by naked eyes. The optimized LAMP method could specifically detect the components of common cattle, yak, buffalo and bison within 1 h, with a detection sensitivity of 0.020 ng·μL-1, and could specifically detect the samples containing beef components in meat products on the market. The LAMP method established in this study had the characteristics of high specificity, high sensitivity, quick response, simple operation and no need of precision instruments, which could be applied to the actual detection of common cattle and their related species in meat products, and provide a strong guarantee for the safety of meat products in China.

Progress on Identification Methods of Animal-derived Ingredients in Meat and Meat Products
JI Yi, CHEN Xiaoyun, DING Lin, WANG Xiaofu, PENG Cheng, XU Xiaoli, XU Junfeng^*
2020, 10(6): 711-716. DOI: 10.19586/j.2095-2341.2020.0136
Abstract ( ) PDF (1109KB)( )

Meat adulteration has a direct impact on human health, public health and safety and social stability, and has become one of the hot topics in food safety. Therefore, it is imperative to detect and identify animal-derived components in meat and meat products efficiently and accurately. Based on this, this paper mainly introduced different research methods for the detection and identification of animal-derived components, analyzed their advantages and disadvantages, and looked forward to the research and development direction of identification methods of animal-derived components in meat and meat products, in order to provide data reference for this field.

Research and Development of Multiplex Real-time Fluorescence PCR Detection Method for Live Pathogenic Bacteria in Food and Beverage
KE Zhenhua
2020, 10(6): 717-727. DOI: 10.19586/j.2095-2341.2020.0107
Abstract ( ) PDF (9764KB)( )

In order to meet the needs of rapid detection of pathogens such as food and beverage, and to establish a pathogen screening method, we selected Shigella dysenteriae, Staphylococcus aureus, Vibrio parahaemolyticus, Enterobacter cloacae, Enterobacter aerogenes, Salmonella, Bacillus cereus, Escherichia coli O157∶H7 and Listeria monocytogenes to carry out multiplex real-time fluorescence PCR methods. To save time and improve efficiency, we developed a general medium for several kinds of bacteria and extracted nucleic acid by high temperature pyrolysis. Screening live bacteria DNA and inactivated dead bacteria by PMA dyes, and detected target bacteria by multiplex real-time fluorescence PCR technology. The detection of target bacteria can be completed within 16 h. The detection limit of target pathogen can reach 103 CFU mL-1.

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