In this study, the molecular marker technology of SRAP and ISSR were applied in rapid identification of seeds from eight species of Brassica oleracea L. Firstly, using the genomic DNA of cabbage as template, SRAP and ISSR reaction systems were optimized through testing every factor, respectively, that affects PCR amplification. Then, using the optimized reaction systems, 30 SRAP primer pairs and 15 ISSR primers were applied to amplify genomic DNA of cabbage, savoy, purple cabbage, borecole, cauliflower, broccoli, Brussels sprouts, and kohlrabi The results showed that high polymorphisms were exhibited among the eight species of Brassica oleracea L. by SRAP primer pairs of M3-E5 and M4-E5, as well as ISSR primers of 844 and 888, especially primer 844 which can identify all eight materials efficiently.
Brassica ; genetics ; Nucleic Acid Amplification Techniques ; methods ; Polymorphism, Genetic ; Repetitive Sequences, Nucleic Acid ; Seeds ; genetics

Multiple displacement amplification (MDA) is a new technology for whole genome amplification (WGA), which can generate large amount of high-quality DNA and features high amplification efficiency and fidelity. MDA combined with conventional PCR techniques has been successfully applied for preimplantation genetic diagnosis, which has broaden latter's clinical applications.
Genome, Human ; Humans ; Nucleic Acid Amplification Techniques ; methods ; Polymerase Chain Reaction ; methods ; Preimplantation Diagnosis ; methods

OBJECTIVE: To establish a method for whole genome amplification (WGA) based on (multiple displacement amplification MDA), and achieve DNA analysis of the human genome from low copy number (LCN). METHODS: DNA sample was used for WGA according to the REPLI-g kit protocol (QIAGEN, Germany). WGA product was used for DNA analysis according to the Applied Biosystems Profiler Plus kit protocol (ABI, USA). RESULTS: WGA product of DNA sample (10 pg) can be used for STR genotyping. CONCLUSION WGA technology could be helpful for LCN DNA analysis.
DNA/analysis* ; DNA Fingerprinting/methods* ; Genome, Human ; Genotype ; Humans ; Microsatellite Repeats ; Nucleic Acid Amplification Techniques/methods*

OBJECTIVETo develop a loop-mediated isothermal amplification (LAMP) method for rapidly diagnosing of Escherichia coli (EHEC) O157:H7 in pathogen detection department or small-scale laboratories.

METHODSPrimers for LAMP test were designed by targeting the antigen coding rfbE of EHEC O157:H7, the Shiga-like toxin stx2 and the fliC encoding gene of H7 flagella antigen, respectively. The reaction condition and reaction system of LAMP were optimized. 2 EHEC O157:H7 type strains, 17 local strains and 33 other enterobacteria were analyzed to evaluate the LAMP's specificity and sensitivity. The results of the LAMP reaction were also compared with routine PCR method.

RESULTSThe amplification products of O157 which had the corresponding target genes turned green by visual inspection and had ladder-like pattern on the gel, but products of other enterobacteria remained orange by visual examination and had no band on the gel. The detection results of LAMP were the same as of routine PCR method. The reaction time of the LAMP method was only 1.5 hours and the detection limit of LAMP assay was 26 CFU/reaction. In addition, the LAMP results could be determined only by visual inspection.

CONCLUSIONLAMP assay is rapid, specific, and sensitive for the detection of EHEC O157:H7. This method might not only reduce the dependence of complicated equipments but also be a potential method for wider use in pathogen detection department, small-scale laboratory, emergency motor vehicle or field survey.

OBJECTIVE To assess the application value of multiplex ligation-dependent probe amplification (MLPA) for the detection of gene deletion and prenatal diagnosis of α-thalassemia. METHODS MLPA was applied for 2 cases with α-thalassemia phenotype by whole blood cell counting and hemoglobin component detection but were ruled out by regular molecular diagnosis. Potential gene deletions and point mutations of α-thalassemia gene were detected with regular Gap-polymerase chain reaction (Gap-PCR) and reverse dot blotting (RDB) in 89 cases where one or both partners were carriers of α-thalassemia mutations. Meanwhile, MLPA was used for detecting α-globin gene deletion among the 89 samples. RESULTS For the 2 cases with α-thalassemia phenotype, no α globin gene deletion was detected by MLPA, but were subsequently confirmed as iron-deficiency anemia. The results of MLPA and Gap-PCR detection for the 88 cases were consistent, except for 1 fetal sample (chorionic villi) which could not be diagnosed by Gap-PCR and was confirmed to be - SEA/αα by MLPA. CONCLUSION MLPA can be applied to prenatal diagnosis of α-thalassemia as an effective supplement to Gap-PCR to reduce both misdiagnosis and missed diagnosis and improve the accuracy of prenatal diagnosis.
Adult ; Female ; Humans ; Nucleic Acid Amplification Techniques ; methods ; Pregnancy ; Prenatal Diagnosis ; methods ; alpha-Thalassemia ; diagnosis ; genetics

Objective: To establish an ultra-sensitive, ultra-fast, visible detection method for Vibrio parahaemolyticus (VP) . Methods: We established a new method for detecting the tdh and trh genes of VP using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 12a (CRISPR/Cas12a) combined with recombinase polymerase amplification and visual detection (CRISPR/Cas12a-VD). Results: CRISPR/Cas12a-VD accurately detected target DNA at concentrations as low as 10 ^-18 M (single molecule detection) within 30 min without cross-reactivity against other bacteria. When detecting pure cultures of VP, the consistency of results reached 100% compared with real-time PCR. The method accurately analysed pure cultures and spiked shrimp samples at concentrations as low as 10 ² CFU/g. Conclusion The novel CRISPR/Cas12a-VD method for detecting VP performed better than traditional detection methods, such as real-time PCR, and has great potential for preventing the spread of pathogens.
CRISPR-Cas Systems ; Nucleic Acid Amplification Techniques/methods* ; Recombinases/genetics* ; Vibrio parahaemolyticus/genetics*

A molecular diagnostic assay which could be stored at room temperature was developed to rapidly detect Mycobacterium tuberculosis (MTB) based on loop-mediated isothermal amplification (LAMP) technology and dry-reagent process. LAMP uses 4 or 6 primers and Bst DNA polymerase to amplify DNA at a constant temperature. The results showed that the LAMP assay could detect the amplification of IS6110 target gene within 20 min using real-time fluorescence signal detection. The sensitive of LAMP assay was similar to the PCR technology while the precision of PCR was better than LAMP (coefficient of variation, LAMP 18.9%, PCR 3.4%), meaning LAMP was more suitable for qualitative detection. The LAMP assay did not amplify DNA of other 10 types of pathogens, including Neisseria meningitidis, Haemophilus influenzae, Staphylococcus aureus, Streptococcus pneumoniae, Rubivirus, mumps virus, adenovirus (type 3), adenovirus (type 7), respiratory syncytial virus B and parainfluenza virus type 2, indicating a good specificity. Furthermore, a dry-reagent assay was developed using air-drying and freeze-drying process. The performance of dried reagents did not change after 10 days storage at 50 ℃, meaning the dried reagents could be stored at room temperature (25 ℃) for more than six months. The dry-reagent LAMP assay also successfully amplified MTB DNA from several clinical samples within 20 min. In conclusion, the developed LAMP assay together with isothermal amplifier could rapidly detection MTB.
Humans ; Mycobacterium tuberculosis/genetics* ; Indicators and Reagents ; Sensitivity and Specificity ; Nucleic Acid Amplification Techniques/methods* ; DNA

A molecular diagnostic assay which could be stored at room temperature was developed to rapidly detect Mycobacterium tuberculosis (MTB) based on loop-mediated isothermal amplification (LAMP) technology and dry-reagent process. LAMP uses 4 or 6 primers and Bst DNA polymerase to amplify DNA at a constant temperature. The results showed that the LAMP assay could detect the amplification of IS6110 target gene within 20 min using real-time fluorescence signal detection. The sensitive of LAMP assay was similar to the PCR technology while the precision of PCR was better than LAMP (coefficient of variation, LAMP 18.9%, PCR 3.4%), meaning LAMP was more suitable for qualitative detection. The LAMP assay did not amplify DNA of other 10 types of pathogens, including Neisseria meningitidis, Haemophilus influenzae, Staphylococcus aureus, Streptococcus pneumoniae, Rubivirus, mumps virus, adenovirus (type 3), adenovirus (type 7), respiratory syncytial virus B and parainfluenza virus type 2, indicating a good specificity. Furthermore, a dry-reagent assay was developed using air-drying and freeze-drying process. The performance of dried reagents did not change after 10 days storage at 50 ℃, meaning the dried reagents could be stored at room temperature (25 ℃) for more than six months. The dry-reagent LAMP assay also successfully amplified MTB DNA from several clinical samples within 20 min. In conclusion, the developed LAMP assay together with isothermal amplifier could rapidly detection MTB.
Humans ; Mycobacterium tuberculosis/genetics* ; Indicators and Reagents ; Sensitivity and Specificity ; Nucleic Acid Amplification Techniques/methods* ; DNA

OBJECTIVES: To explore the ability of inhibition resistibility of multiple displacement amplification （MDA） in samples with inhibitors. To explain the application and value of MDA in forensic medicine by comparing with using magnetic beads methods （MBM） to purify sample. METHODS: Different concentrations of hemoglobin and humid acid （HA） mixed with DNA samples and then divided the samples into MDA group, MBM group and control group. D3S1358 locus was amplified and detected by polyacrylamide gel electrophoresis detection system and AmpFℓSTR® Identifiler™ Plus Kit-capillary electrophoresis detection system. RESULTS: When hemoglobin concentrations exceed 1 ng/μL or HA concentrations exceed 0.1 ng/μL, amplification products could not be obtained by single-locus system in control group. When hemoglobin concentration exceeds 100 ng/μL or HA concentrations exceed 1 ng/μL, the samples could not be amplified by MBM. Inhibitors in different concentrations were amplified successfully in MDA group without any influence from inhibitors. CONCLUSIONS MDA has the capability to remove the inhibition of hemoglobin and HA, which is better than MBM and has a certain value in forensic practices.
DNA/analysis* ; Electrophoresis, Polyacrylamide Gel ; Forensic Medicine/methods* ; Hemoglobins ; Humans ; Humic Substances ; Nucleic Acid Amplification Techniques

Objective To evaluate the efficiency of REPLI-g® Single Cell Kit for sample DNA amplification, and explore its application value in forensic trace DNA amplification. Methods Three DNA extraction kits were selected to extract DNA from peripheral blood of 10 unrelated individuals. The DNA yield and purity of the three DNA extraction kits were compared. According to the results of comparison, one DNA sample was selected to concentrate and dilute, then used as the initial sample of whole genome amplification （WGA）. REPLI-g® Single Cell Kit was used to amplify the initial sample at the whole genome level. The amplification yield and amplification times were calculated, and the distribution of DNA fragments was detected by agarose gel electrophoresis. Goldeneye® DNA ID System 20A Kit was used to perform the STR typing of the initial sample and DNA samples amplified at the whole genome level to evaluate the performance of REPLI-g® Single Cell Kit in trace DNA amplication in terms of purity and yield as well as the success rate of STR typing. Results After comparison, one DNA sample was selected from QIAsymphony® DNA Investigator® Kit extracts to concentrate and dilute as the initial sample of WGA. After amplifying the whole genome of a series of initial samples by REPLI-g® Single Cell Kit, the lowest average of amplification yield reached 8.77×103 ng, while the average of the corresponding amplification times reached 1.40×106. DNA fragments were large and concentrated. The STR typing success rate of WGA samples became lower with the decrease of initial samples used, but when the amount of samples was lower than 0.5 ng, the STR typing success rate of samples after DNA WGA was higher than that of samples without DNA WGA. Conclusion REPLI-g® Single Cell Kit can increase the yield of template DNA. Especially for trace DNA, the STR typing success rate can be improved to a certain extent.
DNA ; DNA Fingerprinting ; Humans ; Microsatellite Repeats ; Nucleic Acid Amplification Techniques/standards* ; Sequence Analysis, DNA/methods*