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@#Bioluminescence is a widespread phenomenon in nature, and luminescent organisms can be found both on land and in the ocean. Among them, luciferase based bioluminescence systems have been widely studied, inspiring the exploration of genetic and epigenetic aspects and the development of a series of related assays for in vivo and in vitro studies. This paper summarizes the recent developments of luciferase based bioluminescence assays in terms of bioluminescence systems, types of luciferases, and the development and application of luciferase bioluminescence assays.
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@#Gene editing tools with nucleases as the main component have now implemented programmable targeted mutagenesis or insertion or deletion of mammalian genomes.From zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR/Cas system to safer and more accurate Cas9 fusion protein gene editing tools and other nuclease gene editing tools, this paper systematically describes the development and evolution of gene editing, with detailed introduction to the development and optimization of next-generation gene editing tools, and a prospect of the clinical application of and challenges for gene editing tools.
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@#The corona virus disease 2019 (COVID-19) caused by the new coronavirus (SARS-CoV-2) has spread rapidly around the world,posing a serious threat to the public"s health. As of September 30,2020,the number of infected people in the world has reached 33 million,causing more than 1 million deaths. Normalized nucleic acid detection methods based on lab have long turnaround time and high cost. Therefore,there is an urgent need to develop a convenient method to detect SARS-CoV-2,so as to achieve rapid testing and timely control of the epidemic when resources are limited.This review summarizes the point-of-care testing (POCT) methods developed for SARS-CoV-2 in terms of extraction,amplification and detection,and briefly introduces commercial POCT instruments that integrate these three steps,in order to provide references for emergency response and rapid deployment of COVID-19 and other emerging infectious diseases.
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Objective To investigate the curative effect of Zhizhukuanzhong capsule combined with Deanxit in the treatment of functional dyspepsia ( FD) of spleen -deficiency and qi -stagnation.Methods Seventy -six patients with FD were randomly divided into three groups .A group (24 cases)_was given Zhizhukuanzhong combined with Deanxit,B group (26 cases) was given Zhizhukuanzhong ,C group (26 cases) received trimebutine dispersible tablets treatment.After treatment for 8 weeks,the symptoms of indigestion,depressive state and therapeutic effect were observed.Results Before treatment,there were no statistically significant differences in the symptoms of dyspepsia (all P>0.05).After treatment,the symptoms of dyspepsia were improved in different degree ,the effective rates of A, B,C three groups were 91.6%,65.4% and 42.3%,respectively,there was no statistically significant difference between A group and B group(χ2 =1.579,P=0.209);there was statistically significant difference between A group and C group(χ2 =13.549,P=0.000);there was no statistically significant difference between B group and C group (χ2 =2.786,P=0.09).The FD patients complicated with varying degrees of anxiety and depression ,there was no statistically significant difference among the three groups before treatment (P>0.05).After treatment,comparison of HAMA and HAMD scores among the three groups:there were statistically significant differences between A group and B group (tHAMA =6.839,tHAMD =4.607,all P<0.05),A group and C group (tHAMA =20.069,tHAMD =15.342,all P<0.01),B group and C group (tHAMA =11.951,tHAMD =12.071,all P<0.01).After treatment,the HAMA,HAMD scores of A group and B group were significantly decreased compared with before treatment ( A group tHAMA =52.758,tHAMD=49.970,B group tHAMA =30.230,tHAMD =17.151,all P<0.01).Those in C group had no statistically significant decline compared with before treatment (tHAMA =7.845,tHAMD =3.530,all P>0.05).The adverse reactions of A group were mainly weakness ,dizziness,drowsiness,which were alleviated within 2 weeks.The adverse reactions of B group were slight stomach disease and stool frequency increased ,which not affected the treatment.The adverse reactions of C group were digestive tract symptoms ,which lasted for more than 2 weeks.Conclusion Zhizhukuanzhong capsule combined with Deanxit in the treatment of FD of spleen -deficiency and qi -stagnation can obtain good curative effect,it is safe,convenient,and with minor side effects,good compliance.
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Early detection and treatment of high-risk adenomas and colorectal cancer (CRC) can reduce mortality of this disease.CRC screening is aimed at minimizing its harm and colonoscopy is presently the gold standard for it.However,colonoscopy needs bowel preparation and is invasive with high risk of intestinal perforation,causing a bad compliance,which is unfavorable to its popularization and application.Recently,non-invasive detection methods for CRC have gone through a rapid development.Tests based on CRC-related biomarkers in fecal and blood samples provide new options for non-invasive CRC screening.However,detection methods for these biomarkers still need further research and improvement because of the complex composition of feces and blood.In the two aspects of fecal tests and blood tests,the progress of recent studies on non-invasive screening methods for CRC was reviewed in this article.
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Objective To evaluate the curative effect of valproate and levetiracetam in children with epilepsy.Methods32 patients in the first people's hospital of Xiaoshan district from March 2015 to November 2016 application of levetiracetam in treatment of children with epilepsy as the study group, another year over the same period in 35 cases of infantile epilepsy in children in our hospital using sodium valproate treatment as the observation group, two groups of children with different drugs in the treatment of the obtained to compare therapeutic effect.ResultsCompared with the control group (71.9%), the effective rate of the treatment group was significantly higher than that of the control group (P<0.05), and the effective rate was 85.7%.ConclusionSodium valproate is an ideal drug for the treatment of epilepsy in children, the two drugs have a certain anti epileptic effect, but compared to the effect of sodium valproate in the treatment of clinical advantages.
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Tacrolimus exhibits varied individual pharmacokinetic and a narrow therapeutic window, resulting in difficulties in personalized medication.In order to improve the safety of tacrolimus in clinical application and its efficiency and rationality in clinical practice, many countries and regions in the world have issued a number of guidelines for tacrolimus application.However, these guidelines generally aim at particular disease and race, and have certain limitation.In this article, the guidelines were explicated and analyzed in detail.Moreover, an individual tacrolimus medication recommendation for Chinese population was summarized based on the latest research of tacrolimus pharmacogenomics and therapeutic drug monitoring so as to provide assistance for the rational use of tacrolimus.
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Objective To study the influence of the minimal invasive Nuss procedure on the pulmonary function of post-operative pectus excavatum patients. Methods Conduct retrospective analysis on the data from 676 pectus excavatum patients who were treated by the minimal invasive Nuss procedure from August 2006 to November 2014. Wherein 182 cases have com-plete preoperative and postoperative pulmonary-function data of one year, three years. These cases were divided into 3 groups according to the age, namely, children group( from 6 to 12 years old, 34 cases) , adolescents group( from 13 to 18 years old, 80 cases) , adults group( above 18 years old, 68 cases) , among which there were 71 cases with pulmonary function data of 1 year after removal of steel plate, they were divided into 3 groups in the same way,namely, children group(20 cases), adoles-cents group(22 cases), adults group(29 cases) . To compare and analyze the pulmonary function indicatrix of patients with dif-ferent ages in preoperative stage, 1 year, 3 years postoperative stages and 1 year after dismantling the steel plate stage, and to investigate the influence of the minimal invasive Nuss procedure on the postoperative lung function. Results The pulmonary function indicatrix in preoperative stage, 1 year, 3 years postoperative stages of the children group did not have significant difference(P>0. 05); the FVC, FEV1 indicatrix of adolescents and adults groups declined after operation in 1 year and 3 years compared with the preoperative stage(P<0. 05), FEF 25％ -75％, FEF 50％, FEF75％ were improved after operation in 1 year and 3 years compared with the preoperative stage(P<0. 05);the pulmonary function indicatrix of three age groups in the 1 year after dismantling the steel plate stage had all improved, in which the pulmonary function indicatrix of the children group improves most significantly(P<0. 05). Conclusion After the minimal invasive Nuss procedure before the plate dis-mantling process, the pulmonary function of children patients remains to be similar. Partial ventilatory function was damaged in the adolescents and adults patients. After the plate dismantling process, the pulmonary function indicatrix of each age group hasimproved in different degrees. Improvement effect is the most significant in patients below the age of 12.
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There are significant individual differences in the antiplatelet effects of aspirin.Three single nucleotide polymorphisms (SNPs),rs5918,rs12041331 and rs730012,are reported to significantly correlate with the efficacy and side effects of aspirin.In the present study,the genotyping method of the three SNPs was established based on the combination of polymerase chain reaction and pyrosequencing technology.Amplification and sequencing primers were designed independently;the amplification conditions were optimized to amplify the three SNPs in the same condition.The sensitivity of the method was detected using original genome DNA at different concentrations.In order to testify the accuracy of the method,the proposed method and Sanger sequencing technology were both used to genotype the three SNPs in 20 blood samples.The results demonstrated that the genotyping method of aspirin-related SNPs was successfully established,with the detection limit being as low as 0.4 ng genome DNA.The genotype results of 20 samples by the proposed method were exactly the same as that of Sanger sequencing.It is evident that the proposed method is sensitive and accurate.
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Objective The NDRG4 gene methylation in stool is a candidate biomarker for non?invasive diagnosis of colorectal cancer. However, the traditional methods for methylation detection could not be well applied to stool samples due to the low sensitivity and low specificity. The aim of this study was to develop a highly sensitive and specific method for quantifying the methylated NDRG4 gene in stools. Methods Forty one stool samples were collected from 12 colorectal cancer patients, 4 adenoma patients and 25 nor?mal persons. The invasive reaction was combined with real?time PCR and the relative quantification was performed by 2-ΔCT method to develop the highly sensitive and specific methylated DNA detection method, which was used for detecting NDRG4 methylation levels in 41 of stool samples. Results The sensitivity of the method was as low as 10 copies of methylated NDRG4 gene fragments. The specificity was high enough to distinguish 0.01% of methylated fragments from un?methylated fragments and 105 copies of unmethylated NDRG4 fragments gave noamplification signals. The detection results from 41 of stool samples showed that detection rate of the NDRG4 gene in stool from adenoma and colorectal cancer groups had a significant difference comparing to that from the normal group. Conclusion The 2-ΔCT method could accurately quantify the methylation levels of the NDRG4 gene in stool samples, and provide an efficient tool for non?invasive colorectal cancer detection.
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Flap endonuclease 1 (FEN1) is an endonuclease that catalyzes invasive reaction. It can be used in signal-amplification reaction-based nucleic acid assay. However, the application of FEN1 is hampered due to the lack of detailed protocols to express and purify the enzyme, and to quantify the enzyme activity. In this paper, the DNA fragment coding the gene of FEN1 from Archaeoglobus fulgidus was synthesized, and inserted into the plasmid of pET24a(+) to express recombinant FEN1 with His-tag. After optimizing the expression, detailed expression protocol of FEN1 was obtained by culturing the recombinant E. coli at 37 ℃ with 200 r/min of shaking for 8 h, followed by inducing with 0.05 mmol/L IPTG at 37 ℃ for 11 h. The purified recombinant FEN1 with the molecular mass of 38 kDa was obtained by Ni-affinity chromatography. Moreover, we developed a accurate quantification method with fluorescence-labelled probes. Finally, the recombinant FEN1 was used in real-time PCR coupled with high specific invader assay for aldh2 gene genotyping to obtain the correct typing results, indicating that the recombinant FEN1 can be used in gene polymorphism detection. We provide a reliable enzyme for developing invasive reaction-based nucleic acid assay.
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A method for the real-time polymerase chain reaction ( PCR ) coupled with high specific invader assay to detect single nucleotide polymorphism ( SNP) was established. To reduce the background signal, the amount of flap endonuclease 1 ( FEN1 enzyme ) and wild-type detection probe was optimized. Under the optimum conditions including 0. 05 μmo/L invasive oligonucleotide probe, 0. 125 μmol/L wild-type detection probe, 0. 5 μmol/L mutation detection probe, 0. 25 μmol/L each fluorescence resonance energy transfer (FRET) probe and 1. 5 U FEN1, the background signal of wild-type sample and mutation sample was dramatically decreased and the background interference to the detecting results was thus eliminated. A total of 21 cases of aldehyde dehydrogenase-2*2 ( ALDH2*2 ) , 19 cases of cytochrome p450 2 C19*2 ( CYP2 C19*2 ) and 19 cases of CYP2C19*3 were analyzed with the established method, and the genotypes of ALDH2*2 were 10 cases of GG homozygote, 8 cases of GA heterozygote and 3 cases of AA homozygote; the genotypes of CYP2C19*2 were 9 cases of GG homozygote, 8 cases of GA heterozygote and 2 cases of AA homozygote;and the genotypes of CYP2C19*3 were 18 cases of GG homozygote and 1 case of GA heterozygote. These results were consistent with those by pyrosequencing. The established method was specific, simple, short time-consuming and low cost, and could be used for the detection of SNP genotyping with non-polluting in single closed tube.
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Proteins presence and differences of the expression level can clarify the physiological or pathological changes in organisms;so the quantitative detection of proteins is vital for disease mechanism research;diagnosis and prognosis evaluation.Traditional protein quantitation methods at the tissue level reflected the average protein expression in cells;but ignore the differences between individual cells.In contrast;approaches for quantitative detection at single-cell level can better reflect the differences.Recently;a number of approaches for such detec-tion have been proposed;including microfluidics;microwell-based technology;optical fiber nanobiosensor;activity-based probe technology and mass spectrometry.The principles;advantages and drawbacks of these approaches are briefly introduced in this review.
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Pyrosequencing is one of the important genetic polymorphism detection methods currently, but the complicated pretreatment procedure limits its application in clinical test. To simplify the whole process of pyrosequencing, on the basis of the linear_after_the_exponential_polymerase chain reaction ( LATE_PCR) , we improved the primer design method of LATE_PCR, increased the length and the concentration of the excess primer, applied direct amplification technology with whole blood, and established a whole blood_imLATE_PCR method based on common rTaq polymerase and “HpH Buffer” ( High pH buffer ) . The amplification system was optimized, and the influences of blood anticoagulant and the amount of whole blood template were investigated. The single stranded template for the pyrosequencing was obtained by PCR amplification using a single tube in one_step process, and the alcohol dehydrogenase gene polymorphisms of 24 clinical blood samples were then detected successfully. The results could be used to guide clinical individualized medication. The genotypes of ADH1B locus of 24 samples were 6 cases of AA homozygote, 14 cases of AG heterozygote, and 4 cases of GG homozygote. The genotypes of ADH1C were 20 cases of GG homozygote, 4 cases of AG heterozygote, and no cases of AA homozygote.
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Noninvasive early diagnosis of colorectal cancer is conducive for patients to reduce the mortality rate and their suffering from diagnosis procedures.One of the most significant methods to proceed noninvasive early diagnosis of colorectal cancer is analysis of stool DNA.Extracting high quality and great quantity of human genomic DNA from stools guarantees diagnosis accuracy.However,the complexity of stool component hinders DNA extraction.Hence,it is crucial to develop highly efficient extraction methods of human genomic DNA from stools for the DNA analysis-based early diagnosis of colorectal cancer.Currently,two kinds of extraction strategies are employed:one is to directly extract total DNA from stools,the other is to enrich exfoliated colonocytes in stools before DNA extraction.This article reviews the advances on these two kinds of extraction techniques and summarizes their applications.
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Ahigh-resolutionmethodforhumanleukocyteantigen-B(HLA-B)genotypingwasestablished based on the optimized polymerase chain reaction, cloning and sequencing technology. The exon2 and exon3 of HLA-B gene were amplified with primers based on the HLA-B gene sequence. These produced heterozygous alleles were effectively cloned into plasmid DNA based on the principle of plasmid incompatibility, and were followed by bacterial culture. Then Sanger sequencing was carried out and after analyzing the result by software ClustalX2 and IMTG/HLA database comparison, the HLA-B genotype of the samples was achieved. Seven clinical samples were detected, and the results were consistent with those of PCR-SBT genotyping method. The method was cost-effective, high-resolution and it did not require technical software. The use of universal primers simplified the cumbersome design and optimization process of specific primers.
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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.
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Objective To prepare and identify the monoclonal antibody (mAb) specific to epithelial cell adhesion molecule (EpCAM) and explore the function of the mAb.Methods The EpCAM antigen expressed by the prokaryotic expression systems was used to immunize the BALB/c mice,and then the splenic cells from the mice were fused with the Sp2/0 cells to produce hybridomas secreting specific mAb.The positive clones were screened by the ELISA.The western blot analysis was used to identify the reactivity of the mAb to the antigen.Then the immunohistochemistry staining was used to detect EpCAM expression in the 3 primary colorectal carcinoma tissues.Results Three mAb specific to EpCAM were obtained by ELISA tests.Western blot results indicated that these three kinds of antibodies could react to the EpCAM antigen,but no response to the GST tag.Immunohistochemical staining results identified that these mAb could give positive signals to the primary colorectal carcinoma tissues from patients.Conclusion Three mAb specific to EpCAM are obtained and identified,which contributes to the diagnosis and therapy of the carcinoma in the future.
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Pyrosequencing is a tool based on bioluminescence reaction for real-time analyzing DNA sequences. The sensitivity of pyrosequencing mainly depends on luciferase in reaction mixture. However, the instability of pyrosequencing reagents caused by fragile wild Photinus pyralis luciferase (PpL) in conventional pyrosequencing usually leads to unsatisfied results, which limits the application of pyrosequencing. In order to improve the stability of pyrosequencing reagents, the coding sequences of mutant thermostable Luciola lateralis luciferase (rt-LlL) was synthesized, and inserted into the plasmid of pET28a(+) to express the thermostable rt-LlL with a 6 x His-tag in the N terminal. The purified rt-LlL with the molecular mass of 60 kDa was obtained by Ni-affinity chromatography. The specific activity of rt-LlL was determined as 4.29 x 10(10) RLU/mg. Moreover, the thermostability of rt-LlL was investigated, and the results showed that rt-LlL had activity at 50 degrees C, and remained 90% of activity after incubated at 40 degrees C for 25 min. Finally, rt-LlL was used to substitute commercial Photinus pyralis luciferase in conventional pyrosequencing reagent to get thermostable pyrosequencing reagent. Comparing with conventional pyrosequencing reagent, the thermostable pyrosequencing reagent is more stable, and it's activity would not lose when incubated at 37 degrees C for 1 h. This study laid foundation of establishing reliable and stable pyrosequencing system which would be applied in Point-of-Care Testing.
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Animals , Enzyme Stability , Escherichia coli , Genetics , Metabolism , Fireflies , Luciferases , Genetics , Recombinant Proteins , Genetics , Sequence Analysis, DNA , MethodsABSTRACT
Objective To obtain I50 anti-idiotype antibody and identify its activity in vitro.Methods I50 anti-idiotype (Id) antibody gene was amplified from the template of fuse 5-I50 by PCR to construct a prokaryotic expression vector pET25b-I50. The expression of pET25b-I50 in E. coli BL21(DE3) was induced by isopropylthio-β-D-galactopyranoside (IPTG) and was confirmed by SDS-PAGE and Western blot with Ab1(FC2) monoclonal antibody and an anti-hexahistidine tag antibody. The method of dialysis refolding was used to restore the activity of I50 anti-Id antibody, which was measured by Dot-ELISA and lymphocyte proliferation assay. Results The recombinant vector was successfully constructed and the recombinant protein was successfully expressed and purified with 90% purity. The relative molecular weight of the expressed protein was 15 kD, which was in accordance with expectation. The activity of I50 anti-Id antibody could be restored and could promote the proliferation of lymphocyte in a dose-dependent manner. Conclusion These results suggested that I50 anti-Id protein vaccine is likely an option in the therapy against nasopharyngeal carcinoma in vivo.