Detection methods and devices of visual detection in closed tube recombinase polymerase amplification of hepatitis B virus / 医学研究生学报

ObjectiveThe traditional detection method of recombinase polymerase amplificaton (RPA) is not suitable for rapid field detection due to the complicated operation and other factors. Taking the detection of hepatitis b virus (HBV) nucleic acid as an example, it established a detection method of HBV nucleic acid isothermal amplification based on recombinase polymerase amplificaton (RPA) and designed a matching visual detection device of RPA product.MethodsFirstly, a RPA product detection device was designed, which can be used to collect images by taking photos of mobile phones and visually interpret the detection results. Secondly, RPA primers and probes were designed according to the design of HBV gene conserved sequence. Amplification efficiency of each primer pairs were compared though monitoring the RPA reaction of real-time fluorescence curve to screen the best primers and optimize the optimal reaction conditions. Visual detection sensitivity was investigated by using artificial synthesis of HBV target plasmid, and was investigated the specificity of the method by the detection of synthetic plasmid containing hepatitis c virus (HCV), human immunodeficiency virus, treponema pallidum, influenza virus, human papilloma virus DNA fragment. Thirdly, the feasibility of RPA product visualization detection device was verified by comparing with the real-time fluorescence amplification curve. Finally, RPA visual detection was performed on 20 serum DNA samples detected by real-time fluorescence PCR to evaluate the applicability of this method to the detection of actual clinical samples.ResultsThe visual detection device of RPA product was used to realize the negative and positive signals that could be detected by mobile phone photography and visual observation. The visual detection method of HBV nucleic acid RPA amplification could realize the visual detection of DNA targets as low as 1-10 copies of HBV within 30 min at 39 ℃ and had good specificity. The test results of 20 serum DNA samples were completely consistent with those of the commercially available qPCR kit, which preliminarily verified the practicability of the method and the device.ConclusionCombined the established HBV-RPA amplification system with the RPA product visualized detection device, it would be expected to develop a low-cost rapid visualization screening technology platform for HBV nucleic acid in blood.

Advances of the mode of clinical pharmacy care in the era of precision medicine / 医学研究生学报

Precision medicine has become a new mode of modern medicine, and personalized medication is the important embodiment of clinical application of precision medicine. The advances of life science technologies greatly facilitated precision medicine, and also promoted the shift of the mode of clinical pharmacy care from rational drug use and individualized medication to precision medication. To achieve"one person, one mode" clinical dosage regimens,it is necessary to rely on the supports of advanced life science technologies and precisely analyzing and accurately characterizing the biomarker clusters related to individual differences among patients, pathological differences of disease, and disease progression. This article illustrated the recent advances in the application of pharmacokinetics/pharmacodynamics, omics technology and liquid biopsy to the design of dosage regimen, prediction of therapeutic effect and adverse drug reactions, etc. in the era of precision medicine. Furthermore, the development direction of the new model of clinical pharmaceutical care faced on the precision medicine is prospected.

Interpretation of clopidogrel guidelines for individualized medication / 医学研究生学报

Clopidogrel exhibits variable individual pharmacokinetic, and clopidogrel resistance may occur in some patients which can’t obtain effective platelet inhibition. To prevent the occurrence of cardiovascular adverse events and improve the clinical curative effect, the article explicated and analyzed relevant guidelines and the latest research of clopidogrel detailedly, summarized an individual dose recommendation in pharmacogenomics for the rational use of clopidogrel.

Progress in the personalized medicine using pharmacometabonomics / 药学学报

Pharmacometabonomics, as an emerging branch of system biology, has been increasingly used in personalized medicine and showed broad prospects. By means of metabonomics, the complicated and detailed metabolic profile of the patient is described, thus providing more detailed description of the disease phenotype. With this understanding, response of different individuals to the drugs are predicted or evaluated through inherent genetic information of the individual combined with the environmental factors. As a result, appropriate drugs and dosage are chosen, which greatly promotes the realization of the individualized therapy goals. This article describes the emerging field of pharmacometabonomics, and the research results of personalized medicine based on the pharmacometabonomics in recent years are reviewed in detail.

Establishment of a hydrogel chip for high-throughput detection of Y chromosome microdeletions / 中华男科学杂志

<p><b>OBJECTIVE</b>To establish a high-sensitivity, high-specificity and low-cost hydrogel chip platform for the clinical screening of Y chromosome microdeletions.</p><p><b>METHODS</b>Site-specific extended primers with a common sequence at the 5' end were used for hybridizing with the target. The Cy5-dUTP was incorporated into the products by primer extension, and the products were labeled with fluorescence. Then the extended products were added to the chip for hybridizing with acrylamide-modified common probes immobilized on the chip. After removal of the free Cy5-dUTP by electrophoresis, the signals were obtained by fluorescence scanning. And the detecting conditions of this method were optimized.</p><p><b>RESULTS</b>SY254 of 9 samples was successfully detected with the hydrogel chip. The results showed that 3 were normal and the other 6 with microdeletions (1 female sample as a negative control), which coincided with the results of conventional multiplex PCR-electrophoresis.</p><p><b>CONCLUSION</b>The hydrogel chip platform we established has provided a new technique for the detection of Y chromosome microdeletions, and is beneficial to the diagnosis and treatment of male infertility.</p>

Spaceflight alters the gene expression profile of cervical cancer cells / 癌症

Our previous study revealed that spaceflight induced biological changes in human cervical carcinoma Caski cells. Here, we report that 48A9 cells, which were subcloned from Caski cells, experienced significant growth suppression and exhibited low tumorigenic ability after spaceflight. To further understand the potential mechanism at the transcriptional level, we compared gene expression between 48A9 cells and ground control Caski cells with suppression subtractive hybridization (SSH) and reverse Northern blotting methods, and analyzed the relative gene network and molecular functions with the Ingenuity Pathways Analysis (IPA) program. We found 5 genes, SUB1, SGEF, MALAT-1, MYL6, and MT-CO2, to be up-regulated and identified 3 new cDNAs, termed B4, B5, and C4, in 48A9 cells. In addition, we also identified the two most significant gene networks to indicate the function of these genes using the IPA program. To our knowledge, our results show for the first time that spaceflight can reduce the growth of tumor cells, and we also provide a new model for oncogenesis study.

Quantitative pyrosequencing of heterozygous single nucleotide polymorphisms for rapid diagnosis of Down's syndrome / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish a method to detect Down's syndrome through quantitative pyrosequencing of the heterozygous single nucleotide polymorphisms (SNPs) on the chromosome 21.</p><p><b>METHODS</b>An improved allele-specific-amplification was used to screen heterozygous SNPs on the chromosome 21 from 84 normal samples. Pyrosequencing was used to quantitatively determine the ratio between the two alleles of a heterozygote, and the diagnosis of Down's syndrome was thus carried out based on the ratio.</p><p><b>RESULTS</b>By genotyping 84 genomic DNA samples from normal Chinese population, 6 SNPs with a relatively high level of heterozygosity were screened out. Heterozygote coverage of 92.9% was achieved by using a panel of 6 SNPs on the chromosome 21. Ten clinical samples from Down's syndrome patients were quantitatively determined by pyrosequencing, and 9 samples were accurately diagnosed by comparing the ratio of the two alleles. The pyrosequencing results showed that the ratio of the two alleles were 2:1 or 1:2 for the Down's syndrome patients.</p><p><b>CONCLUSION</b>The method has the advantage of a low cost, simple process, and time-saving operation and could be potentially applicable to the rapid diagnosis of Down's syndrome.</p>

Detection of autoantibodies in the sera of nasopharyngeal carcinoma patients / 中南大学学报(医学版)

OBJECTIVE@#To investigate whether there are autoantibodies to nasopharyngeal carcinoma (NPC) in the sera of patients and to find new NPC biomarkers.@*METHODS@#Cell plate of Epstein Barr virus negative NPC cell line CNE1 was prepared, and difference between 32 NPC patient sera and 54 normal sera was analyzed by ELISA. We extracted the total protein of CNE1, and analyzed whether there were specific proteins to react with NPC sera by Western blot.@*RESULTS@#The average absorbance values of serum antibody in NPC patients (0.904+/-0.032) were significantly higher than those of normal serum antibody absorbance values (0.736+/-0.028) (P< 0.01). The analysis with Western blot showed there were positive bands,and some of these were unanimous bands, but the intensity increased, and some of these were new bands compared with the normal sera. These positive bands may be NPC tumor-associated antigens or NPC tumor-specific antigens.@*CONCLUSION@#Autoantibodies that react with NPC exist in the sera of NPC patients, but they do not react with Epstein-Barr virus. It provides the basis to seek the tumor biomarkers in NPC sera.

Direct multiplex-PCR from whole blood for rapid detection of Y chromosome microdeletions / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish a rapid and simple method to detect Y chromosome microdeletions directly using whole blood as starting material for multiplex-PCR.</p><p><b>METHODS</b>Using a self-prepared pHpH-Bufferq, multiplex-PCR amplification was directly carried out from the anticoagulant whole blood sample without DNA extraction step. Twelve sequence tagged sites (STS), namely SY84, SY86, SY127, SY134, SY124, SY132, SY152, SY157, SY239, SY242, SY254 and SY255, in AZFa, AZFb, and AZFc gene regions were detected in 5 different tubes. In order to ensure the validity of the experiments, sex-determining region Y (SRY) and X-linked or Y-linked zinc finger gene (ZFX/Y) were used as internal controls. Furthermore, conventional PCR using genomic DNA extracted from each blood sample was performed in parallel for evaluating the accuracy of the experiments.</p><p><b>RESULTS</b>A total of 156 male samples were detected, and a normal male sample and a female sample were used as a positive and a negative control respectively. The results showed that 144 samples had no deletion; one sample was AZF-deleted; one sample was AZFb-deleted; seven samples were AZFc-deleted; one sample was both AZFb- and AZFc- deleted; and two samples were all AZFa-, AZFb- and AZFc- deleted. The observed results from two kinds of starting material (whole blood and purified DNA) are completely consistent.</p><p><b>CONCLUSION</b>In our method, PCR amplification was directly carried out from whole blood without any DNA extraction step. So it has the advantages of low cost, simple process, time-saving operation and less cross-contamination. The whole process can be completed within 2 hours. Thus the efficiency of clinical detection is improved greatly.</p>

Expression and purification of ATP sulfurylase from Saccharomyces cerevisias in Escherichia coli and its application in pyrosequencing / 生物工程学报

ATP sulfurylase (ATPS,EC 2.7.7.4) reversibly catalyzes the reaction between ATP and sulfate to produce APS and pyrophosphate (PPi), and has been used in pyrosequencing. The gene coding ATP sulfurylase was amplified from the genomic DNA of Saccharomyces cerevisias (CICC 1202), and cloned into prokaryotic expression plasmid pET28a( + ) to provide a recombinant expression plasmid pET28a( + )-ATPS. Upon IPTG induction, ATP sulfurylase was produced by E. coli BL21 (DE3) harboring the recombinant expression plasmid pET28a( + )-ATPS. The relative molecular weight of recombinant ATP sulfurylase with His tag was about 60 kD. The recombinant ATP sulfurylase with electrophoretic pure grade was obtained only by two purification steps: His * Bind Resin affinity chromatography and ultrafiltration. The specific activity of the purified recombinant ATP sulfurylase was as high as 5.1 x 10(4) u/mg. The successful application of the enzyme in pyrosequencing was also demostrated.

Characterization of N-glycan mapping of bioengineering recombinant erythropoietin by capillary electrophoresis with laser-induced fluorescence / 药学学报

<p><b>AIM</b>N-Glycans in recombinant human erythropoietin (EPO) are essential to in vivo biological activity. This paper is to develop a method for mapping sialyated or asialyated N-glycans of EPO.</p><p><b>METHODS</b>At first, N-glycans linked to asparagines in glycoprotein EPO were released by peptide N-glycosidase F. To map asialyated N-glycans, sialic acid in N-glycans were removed by incubating N-glycans with sialidase. Oligosaccharides were labeled with a sensitive fluorescent dye 8-aminopyrene-1,3,6-trisulfonate (APTS), and all of the labeled oligosaccharides released from EPO were mapped by capillary gel electrophoresis with laser-induced fluorescence. The relationship between N-glycans and bioactivity of EPO was investigated on the basis of N-glycan mapping spectra.</p><p><b>RESULTS</b>N-Glycans of seven different batches of EPO were mapped. Each sample was analysed twice, with and without sialidase treatment. The results showed that N-glycans of each sample were approximately the same. But when the expression vector was different, the types of N-glycans and their relative content were quite different. In case of asialyated N-glycan mapping, the retention time of each oligosaccharide delayed greatly, and most importantly, the resulted sialic acid peak can be used as a quantitative standard to determine sialic acid content in N-glycans of EPO. In addition, the difference of N-glycan mapping was observed when the in vivo biological activity of EPO was different.</p><p><b>CONCLUSION</b>The approach in this article for determining N-glycan mapping can be applied to determine the source of EPO and the difference between each batch. It is also a suitable tool for routinely controlling the inner quality of EPO by coupled with peptide mapping.</p>

P53 gene mutation detection by bioluminometry assay / 药学学报

<p><b>AIM</b>To develop a simple, fast and inexpensive approach as well as an instrument for detection of gene mutation.</p><p><b>METHODS</b>Pyrosequencing based on bioluminometry assay was employed to detect gene mutation. Pyrosequencing is a method of sequencing by synthesis step-by-step using four enzymes, DNA-polymerase, ATP sulfurylase, luciferase and apyrase. The signal was produced by detecting pyrophosphate released during a dNTP incorporation. For mutation detection, a DNA fragment was amplified by PCR at first, followed by a single-stranded DNA preparation. In the second step, a short primer was annealed to the position just before the mutation point. Finally, specific dNTPs were added in terms of the template sequence. The mutation species can be readily determined by the sequence.</p><p><b>RESULTS</b>A new instrument was developed for gene mutation detection by pyrosequencing. To iteratively inject small amount of each dNTP for the sequencing reaction, capillaries were used to connect dNTP reservoirs and the reaction chamber. Each dNTP was delivered by adding a gas pressure on the top of a corresponding dNTP reservoir, by which 0.2 microL of dNTP can be exactly added each time. It was theoretically proved that undesired liquid seep through the capillary did not affect the sequencing reactions in pyrosequencing. In addition, the three possible variants (wildtype, mutant and heterozygote) of a mutant point Cys275Ser in P53 gene exon 8 were determined by pyrosequencing using the instrument. A simple method was also described for rapidly distinguishing the type of a variant.</p><p><b>CONCLUSION</b>The developed method is very simple, and the corresponding instrument is inexpensive and easy to operate, which can be used to detect many types of mutation.</p>