Cellular fibronectin (cFn) is a type of bioactive non-collagen glycoprotein regarded as the main substance used to maintain periodontal attachment. The content of cFn in some specific sites can reflect the progress ...Cellular fibronectin (cFn) is a type of bioactive non-collagen glycoprotein regarded as the main substance used to maintain periodontal attachment. The content of cFn in some specific sites can reflect the progress of periodontitis or peri-implantitis. This study aims to evaluate the expression of cFn messenger RNA (mRNA) in tissues of adult periodontitis and peri-implantitis by real-time fluorescent quantitative polymerase chain reaction (PCR) and to determine its clinical significance. A total of 30 patients were divided into three groups of 10: healthy, adult periodontitis and peri-implantitis. Periodontal tissue biopsies (1 mmx I mmx I mm) from each patient were frozen in liquid nitrogen. Total RNA was extracted from these tissues, and the content, purity and integrity were detected. Specific primers were designed according to the sequence, and the mRNA expression levels of cellular fibronectin were detected by real-time PCR. The purity and integrity of the extracted total RNA were both high, and the specificity of amplified genes was very high with no other pollution. The mRNA expression of cFn in the adult periodontitis group (1.526+0.441) was lower than that in the healthy group (3.253+0.736). However, the mRNA expression of cFn in the peri-implantitis group (3.965+0.537) was significantly higher than that in the healthy group. The difference revealed that although both processes were destructive inflammatory reactions in the periodontium, the pathomechanisms were different and the variation started from the transcription level of the cFn gene.展开更多
AIM: To design, optimize and validate a rapid,internally controlled real-time polymerase chain reaction(RT-PCR) test for herpes simplex virus(HSV) in the diagnosis of necrotizing herpes stromal keratitis.· M...AIM: To design, optimize and validate a rapid,internally controlled real-time polymerase chain reaction(RT-PCR) test for herpes simplex virus(HSV) in the diagnosis of necrotizing herpes stromal keratitis.· METHODS: Tears alone or together with corneal epithelium scrapings from 30 patients(30 eyes)suspected of necrotizing herpes stromal keratitis were tested for HSV DNA by RT-PCR. The samples were collected during the first visit and then on the subsequent 7, 14, 28, 42, and 56 d. The symptoms of the patients were scored before treatment to determine the correlation between HSV concentration in the corneal epithelium scrapings and clinical scores.·RESULTS: The positive rate(46.4%) in the corneal epithelium group before the therapy was significantly higher than that(13.3%) in the tears group(P =0.006).There were 13 positive HSV patients before the therapy,the concentration of HSV DNA in corneal epithelium scrapings group was significantly higher than that in the tears group(paired t-test, P =0.0397). Multilevel mixedeffects model analysis showed that the difference between the corneal epithelium scrapings group and the tears group was statistically significant(P =0.0049). The Spearman rank correlation analysis indicated a positive correlation between the HSV concentration in the corneal epithelium scrapings and clinical scores before the treatment(r =0.844, P〈 0.0001).· CONCLUSION: RT-PCR appears to be a powerful molecular tool for the diagnosis of necrotizing herpes stromal keratitis.展开更多
AIM: To develop a real-time reverse transcriptionpolymerase chain reaction(RT-PCR) assay to genotype rotavirus(G and P) in Alberta from January 2012 to June 2013. METHODS: We developed and validated a different approa...AIM: To develop a real-time reverse transcriptionpolymerase chain reaction(RT-PCR) assay to genotype rotavirus(G and P) in Alberta from January 2012 to June 2013. METHODS: We developed and validated a different approach to perform rotavirus G and P genotyping using a two-step SYBR green RT-PCR(rt-g PCR) by selecting genotype-specific primers of published conventional RT nested PCR(cn RT-PCR) assay and optimizing the amplification conditions. c DNA was first synthesized from total RNA with Super Script? Ⅱ reverse transcriptase kit followed by amplication step using monoplex SYBR green real-time PCR. After the PCR reaction, melting curve analysis was used to determine specific genotype. Sixteen samples previously genotyped using cn RT-PCR were tested using the new assay and the genotyping results were compared as sensitivity analysis. Assay specificity was evaluated by testing other gastroenteritis viruses with the new assay. The amplicon size of each available genotype was determined by gelelectrophoresis and DNA sequences were obtained using Sanger-sequencing method. After validation and optimization, the new assay was used to genotype 122 pediatric clinical stool samples previously tested positive for rotavirus using electron microscopy between January2012 and June 2013.RESULTS: The new rt-g PCR assay was validated and optimized. The assay detected G1 to G4, G9, G12 and P[4] and P[8] that were available as positive controls in our laboratory. A single and clear peak of melting curve was generated for each of specific G and P genotypes with a Tm ranging from 80 ℃ to 82 ℃. The sensitivity of rt-g PCR was comparable to cn RT-PCR with 100% correlation of the 16 samples with known G and P genotypes. No cross reaction was found with other gastroenteritis viruses. Using the new rt-g PCR assay, genotypes were obtained for 121 of the 122 pediatric clinical samples tested positive for rotavirus: G1P[8](42.6%), G2P[4](4.9%), G3P[8](10.7%), G9P[8](10.7%), G9P[4](6.6%), G12P[8](23.0%), and unknown GP[8](0.8%). For the first time, G12 rotavirus strains were found in Alberta and G12 was the second most common genotype during the study period. Gel electrophoresis of all the genotypes showed expected amplicon size for each genotype. The sequence data of the two G12 samples along with other genotypes were blasted in NCBI BLAST or analyzed with Rota C Genotyping tool(http://rotac.regatools.be/). All genotyping results were confirmed to be correct.CONCLUSION: rt-g PCR is a useful tool for the genotyping and characterization of rotavirus. Monitoring of rotavirus genotypes is important for the identification of emerging strains and ongoing evaluation of rotavirus vaccination programs.展开更多
AIM:To investigate the stability of the seven housekeeping genes:beta-actin(ActB),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),18s ribosomal unit 5(18s),cyclophilin A(CycA),hypoxanthine-guanine phosphoribosyl trans...AIM:To investigate the stability of the seven housekeeping genes:beta-actin(ActB),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),18s ribosomal unit 5(18s),cyclophilin A(CycA),hypoxanthine-guanine phosphoribosyl transferase(HPRT),ribosomal protein large P0(36B4)and terminal uridylyl transferase 1(U6)in the diabetic retinal tissue of rat model.METHODS:The expression of these seven genes in rat retinal tissues was determined using real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR)in two groups;normal control rats and streptozotocininduced diabetic rats.The stability analysis of gene expression was investigated using geNorm,NormFinder,BestKeeper,and comparative delta-Ct(ΔCt)algorithms.RESULTS:The 36B4 gene was stably expressed in the retinal tissues of normal control animals;however,it was less stable in diabetic retinas.The 18s gene was expressed consistently in both normal control and diabetic rats’retinal tissue.That this gene was the best reference for data normalisation in RT-qPCR studies that used the retinal tissue of streptozotocin-induced diabetic rats.Furthermore,there was no ideal gene stably expressed for use in all experimental settings.CONCLUSION:Identifying relevant genes is a need for achieving RT-qPCR validity and reliability and must be appropriately achieved based on a specific experimental setting.展开更多
Single pollen grain polymerase chain reaction (PCR) has succeeded in several species, however only limited numbers of pollen grains were involved due to difficulties in pollen isolation and lysis. This has limited i...Single pollen grain polymerase chain reaction (PCR) has succeeded in several species, however only limited numbers of pollen grains were involved due to difficulties in pollen isolation and lysis. This has limited its application in genetic analysis and mapping studies in plants. A high-throughput (HT) procedure for collecting and detecting genetic variation in a large number of individual pollen grains by PCR is reported. The HT procedure involved the collection of individual pollen grains by a pair of special forceps and the lysis of pollen grains in a heated alkali/detergent solution followed by neutralization with a tris-ethylenediamine tetraacetic acid (TE) buffer. These resulting template solutions yielded PCR reactions involving the 5S ribosomal RNA intergenic spacers, randomly amplified polymorphic DNA, and simple sequence repeats markers. Using this procedure, one person with experience could collect and process up to 288 single pollen grain PCR reactions per day. The method worked well on sugarcane, corn, Miscanthus spp., snap bean, sorghum, and tomato. The ability to collect and conduct PCR on individual pollen grains on a large scale offers a new approach to genetic analyses and mapping studies in an easily controllable environment with a considerable cost reduction. The method will also significantly benefit studies in species that are difficult subjects for classical genetic research.展开更多
Burkholderia glumae causing seedling rot and grain rot of rice was listed as a plant quarantine disease of China in 2007. It's quite necessary to set up effective detection methods for the pathogen to manage further ...Burkholderia glumae causing seedling rot and grain rot of rice was listed as a plant quarantine disease of China in 2007. It's quite necessary to set up effective detection methods for the pathogen to manage further dispersal of this disease. The present study combined the real-time PCR method with classical PCR to increase the detecting efficiency, and to develop an accurate, rapid and sensitive method to detect the pathogen in the seed quarantine for effective management of the disease. The results showed that all the tested strains of B. glumae produced about 139 bp specific fragments by the real-time PCR and the general PCR methods, while others showed negative PCR result. The bacteria could be detected at the concentrations of 1×10^4 CFU/mL by general PCR method and at the concentrations below 100 CFU/mL by real-time fluorescence PCR method. B. glumae could be detected when the inoculated and healthy seeds were mixed with a proportion of 1:100.展开更多
Background The diagnosis of Pneumocystis pneumonia (PCP) in immunocompromised patients is still challenging today due to the absence of an in vitro culture system and the low diagnostic accuracy of microscopic exami...Background The diagnosis of Pneumocystis pneumonia (PCP) in immunocompromised patients is still challenging today due to the absence of an in vitro culture system and the low diagnostic accuracy of microscopic examinations. Herein, we performed a meta-analysis to evaluate the accuracy of real-time polymerase chain reaction (PCR) in the diagnosis of PCP. Methods We searched Web of Knowledge and Medline from 1990 to May 2010 for studies reporting diagnostic accuracy data regarding the use of real-time PCR in the diagnosis of PCP in immunocompromised patients. Results Ten individual studies were included. Overall, the sensitivity of real-time PCR was 97% (95% CI: 93%-99%); the specificity was 94% (95% CI: 90%-96%). The area under the HSROC curve (95% CO for real-time PCR was 0.99 (0.97-0.99). In a subgroup analysis regarding studies involving HIV patients among the study population, the sensitivity and specificity were 97% (95% CI: 93%-99%) and 93% (95% CI: 89%-96%), respectively. Regarding studies using Bronchoalveolar lavage (BAL) samples only: sensitivity =98% (95% CI: 94%-99%); specificity =93% (95% CI: 89%- 96%), respectively. Regarding studies using microscopy as a reference standard: sensitivity =97% (95% CI: 92%-99%); specificity =93% (95% CI: 88%-96%). However, high between-study statistical heterogeneity was observed in all analyses. Conclusions Real-time PCR has a good diagnostic accuracy and may provide a useful adjunctive tool for the diagnosis of PCP in immunocompromised patients. Further studies are needed in order to identify any differences in the diagnostic performance of real-time PCR in HIV and non-HIV immunocompromised patients.展开更多
Multiplex polymerase chain reaction (PCR) has been widely used to detect Y-chromosome micredeletions, which is one of the major causes of male infertility. Both the European Academy of Andrology (EAA) and the Euro...Multiplex polymerase chain reaction (PCR) has been widely used to detect Y-chromosome micredeletions, which is one of the major causes of male infertility. Both the European Academy of Andrology (EAA) and the European Molecular Genetics Quality Network (EMQN) have recommended the use of sY84 and sY86 markers for the detection of azoospermia factor a (AZFa) microdeletion during DNA testing for male infertility. In this study, a large-scale analysis of AZF microdeletion in a total of 630 Chinese males, including healthy semen donors (n=200), infertile males with normal sperm count (n=226) and patients with either nonobstructive azoospermia or severe oligozoospermia (n=204), was performed. A series of nine sequence-tagged site (STS) markers from the AZF region of the Y chromosome was used to detect microdeletions. All primers were designed based on the recommendations of the National Center for Biotechnology Information. An unusually high incidence (73/630, 11.6%) of sY84-absent but sY86-present genotypes was observed in the AZFa microdeletion screening. Sequencing the sY84-flanking region revealed a total of 73 patients with sY84-absent but sY86-present genotypes have a T-to-G transversion at the fifth base from the 5' end of the reverse sY84 primer. These prevalent false positives, which were not only observed in infertile men, but also observed in donors, resulted from a single-nucleotide polymorphism (SNP) named rs72609647 in the targeting sequence of the reverse sY84 primer. Our study suggests that a pre-screening of existence of rs72609647 polymorphism can prevent the frequent false positive results of AZFa microdeletions detection in the infertile Chinese males. Given the SNP rs72609647 was recently found in a deep sequencing of a Chinese individual, the current EAA and EMQN standards may need to be scrutinized among different populations to avoid the potential genetic variations in the primer binding sequences.展开更多
AIM:To investigate the relationship between c.343A>G and c.2216A>C polymorphism sites in the CDH17 gene and colorectal carcinoma.METHODS:Ninety-three non-consanguineous colorectal carcinoma patients admitted to ...AIM:To investigate the relationship between c.343A>G and c.2216A>C polymorphism sites in the CDH17 gene and colorectal carcinoma.METHODS:Ninety-three non-consanguineous colorectal carcinoma patients admitted to the Department of Oncology at the First Affiliated Hospital of Zhengzhou University were included in this study.Ninety-three peripheral venous blood samples,of approximately one milliliter from each patient,were collected betweenDecember 2009 and August 2010.The genomic DNA of these peripheral venous blood samples were extracted and purified using a Fermentas Genomic DNA Purification Kit(Fermentas,CA) according to the manufacturer' s protocol.The single nucleotide polymorphisms(SNPs) of the liver-intestine cadherin(CDH17) gene c.343A>G and c.2216A>C were determined by the polymerase chain reaction-single strand conformation polymorphism method(PCR-SSCP) in 93 peripheral venous blood samples from patients suffering with colorectal carcinoma.Typical samples that showed different migration bands in SSCP were confirmed by sequencing.Directed DNA sequencing was used to check the correctness of the genotype results from the PCR-SSCP method.RESULTS:There was a significant association between the c.2216 A>C SNPs of the CDH17 gene and the tumor-node-metastasis(TNM) grade,as well as with lymph node status,in 93 peripheral venous blood samples from colorectal carcinoma patients.The genotype frequencies of A/C,A/A,and C/C were 12.90%,33.33% and 53.76%,respectively.There was a significant correlation between lymph node metastasis,TNM grade,and the genotype distribution(P < 0.05).The C/C genotype raised the risk of lymph node metastasis and the TNM grade.There was a significant difference in the TNM grade and lymph node metastasis between the A/A and C/C genotypes(P = 0.003 and P = 0.013,respectively).Patients with colorectal carcinoma carrying the C allele tended to have a higher risk of lymph node metastasis and have a higher TNM grade.The difference between the TNM grades,as well as the lymph node metastasis of the two alleles,was statistically significant(P < 0.01).CONCLUSION:The SNPs of the CDH17 gene c.2216 A>C might be clinically important in the prognosis of colorectal carcinoma.展开更多
The rRNA genetic locus is found in all prokaryotic organisms, and is highly conservative, although its relatively stable variations are found frequently in different bacteria. The utility of this locus as a taxonomic ...The rRNA genetic locus is found in all prokaryotic organisms, and is highly conservative, although its relatively stable variations are found frequently in different bacteria. The utility of this locus as a taxonomic and phylogenetic tool has been reported widely. This study, aimed at 16S rRNA gene (16S rDNA) and with the help of biomolecular methods, attempted to achieve the goal of rapid identification of common pathogens. In this study, 333 clinical isolated pathogenic bacteria were collected. Two pairs of primers were chosen and labeled with different fluorescent dyes and then used to amplify the genomic DNA extracted from bacteria. The PCR products were then detected by capillary electrophoresis-single strand conformation polymorphism (CE-SSCP). In order to pursue higher resolution and peak-separation effect, a high efficient separating medium, liner polyacrylamidedel (LPA), was put to use in this study. Finally, every bacteria colony generated distinct patterns from each other, which were easily to be used for identification. These results indicated that PCR-CE-SSCP was a rapid identification method for bacterial identification, with the aspects of high efficiency and high precision. Compared with traditional method, this technology is of great utility for clinical use especially for its high sensitivity.展开更多
The single cell isolation technique was used to detect fetal nucleated erythroblasts at a single cell level from the peripheral blood of pregnant women in order to investigate the feasibility of this method for nonin...The single cell isolation technique was used to detect fetal nucleated erythroblasts at a single cell level from the peripheral blood of pregnant women in order to investigate the feasibility of this method for noninvasive prenatal diagnosis. Single fetal nucleated erythroblasts were isolated from the peripheral blood samples from 51 pregnant women by micromanipulation techniques after density gradient centrifugation. Nested polymerase chain reaction method was used to amplify the SRY gene. It was found that the concordance rate of amplification results with real fetal sex was 82.61 %. The sensitivity and specificity were 80 % and 87.50 % respectively. It was suggested that it is feasible and promising in non invasive prenatal diagnosis to detect fetal nucleated erythroblasts at a single cell level by using micromanipulation techniques.展开更多
The genomic DNA is the organism’s various physiological and pathological characteristics of the material base.Single nucleotide polymorphisms(single nucleotide polymorphisms,SNPs) is the genome of the most common for...The genomic DNA is the organism’s various physiological and pathological characteristics of the material base.Single nucleotide polymorphisms(single nucleotide polymorphisms,SNPs) is the genome of the most common form of genetic variation in the human genome widespread.With the completion of the Human Genome Project to stimulate,there is growing focus on research developed in this latest 3rd generation of genetic markers of practical significance.This paper describes the classification of SNPs and characteristics of the human genome SNP s research and development and the present in today’s practice,the application of design experimts to explore the direct sequencing of amplified using PCR-based method showed that SNPs.展开更多
BACKGROUND Head and neck squamous cell carcinoma(HNSCC) is considered to be a progressive disease resulting from alterations in multiple genes regulating cell proliferation and differentiation like receptor tyrosine k...BACKGROUND Head and neck squamous cell carcinoma(HNSCC) is considered to be a progressive disease resulting from alterations in multiple genes regulating cell proliferation and differentiation like receptor tyrosine kinases(RTKs) and members of the fibroblast growth factor receptors(FGFR)-family. Singlenucleotide polymorphism(SNP) Arg388 of the FGFR4 is associated with a reduced overall survival in patients with cancers of various types. We speculate that FGFR4 expression and SNP is associated with worse survival in patients with HSNCC.AIM To investigate the potential clinical significance of FGFR4 Arg388 in the context of tumors arising in HNSCC, a comprehensive analysis of FGFR4 receptor expression and genotype in tumor tissues and correlated results with patients' clinical data in a large cohort of patients with HNSCC was conducted.METHODS Surgical specimens from 284 patients with HNSCC were retrieved from the Institute of Pathology at the Ludwig-Maximilian-University in Germany.Specimens were analyzed using immunohistochemistry and polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP). The expression of FGFR4 was analyzed in 284 surgical specimens of HNSCC using immunohistochemstry. FGFR4 polymorphism was detected by PCR-RFLP.Patients' clinical data with a minimum follow-up of 5 years were statistically evaluated with a special emphasis on survival analysis employing Kaplan-Meier estimator and Cox regression analysis.RESULTS Concerning the invasive tumor areas the intensity of the FGFR4 expression was evaluated in a four-grade system: no expression, low expression, intermediate and high expression. FGFR4 expression was scored as "high"(+++) in 74(26%),"intermediate"(++) in 103(36.3%), and "low"(+) in 107(36.7%) cases. Analyzing the FGFR4 mutation it was found in 96 tumors(33.8%), 84 of them(29.6%) having a heterozygous and 12(4.2%) homozygous mutated Arg388 allele. The overall frequency concerning the mutant alleles demonstrated 65% vs 34% mutated alleles in general. FGFR4 Arg388 was significantly associated with advanced tumor stage(P < 0.004), local metastasis(P < 0.0001) and reduced disease-free survival(P < 0.01). Furthermore, increased expression of FGFR4 correlated significantly with worse overall survival(P < 0.003).CONCLUSION In conclusion, the FGFR4 Arg388 genotype and protein expression of FGFR4 impacts tumor progression in patients with HNSCC and may present a useful target within a multimodal therapeutic intervention.展开更多
Aim To use and modify the molecular histology technique that was first introduced by Hansmann et al and to investigate the cell origin and the clonality of Hodgkin/Reed Sternberg (H/R S) cell. Method Single H/R S cell...Aim To use and modify the molecular histology technique that was first introduced by Hansmann et al and to investigate the cell origin and the clonality of Hodgkin/Reed Sternberg (H/R S) cell. Method Single H/R S cells were isolated by micromanipulation from frozen histological sections of tissues affected by Hodgkins disease. After DNA was extracted from these cells, PCR was performed with primers designed for β globin gene that exists in any somatic cells. Results The results showed that a special PCR product (268bp) was found in 12 out of 38 H/R S cells (positive rate of 31.6% ) with DNA from the cells micropicked from the frozen sections, whereas no any products was found with the DNA from these cells of formalin fixed and paraffin embedded sections. Conclusions The molecular histology technique we used is suitable for detecting the immunoglobulin heavy and light chain gene rearrangement in single H/R S cells.展开更多
The quantitative realtime polymerase chain reaction(RT-qPCR)is a powerful and sensitive method to measure expression of targeted gene but it highly relies on the use of suitable reference genes for data normalization....The quantitative realtime polymerase chain reaction(RT-qPCR)is a powerful and sensitive method to measure expression of targeted gene but it highly relies on the use of suitable reference genes for data normalization.We evaluated the expressions of 8 housekeeping genes:18S ribosomal rDNA(18S rDNA),28S ribosomal r DNA(28S rDNA),rubisco large subunit(rbc-L),β-actin(ACT),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),elongation factor 1(EF1),β-tubulin(Tub B),and P-phycoerythrin B(PEB),to select the suitable reference genes for different life-history stages(tetrasporophyte,carposporophyte,and male/female gametophyte)of Gracilaria vermiculophylla by absolute quantitative method.Softwares geNorm and BestKeeper were used to verify the results acquired from copy number analysis.Results show that the expression of identified reference genes varied in comparing groups composed of different type of life stages.It is suggested that 18S rDNA and TubB could be used for highly complex samples composed of mixed ploidy and phases.18S rDNA and 28S rDNA were also preferred for using among the matured isomorphic samples.But for samples with different maturities,TubB and ACT were recommended for tetrasporophytes and gametophytes respectively.展开更多
基金the National Natural Science Foundation(Project No.81070868/H1409)the State Key Laboratory of Oral Diseases,Sichuan University.
文摘Cellular fibronectin (cFn) is a type of bioactive non-collagen glycoprotein regarded as the main substance used to maintain periodontal attachment. The content of cFn in some specific sites can reflect the progress of periodontitis or peri-implantitis. This study aims to evaluate the expression of cFn messenger RNA (mRNA) in tissues of adult periodontitis and peri-implantitis by real-time fluorescent quantitative polymerase chain reaction (PCR) and to determine its clinical significance. A total of 30 patients were divided into three groups of 10: healthy, adult periodontitis and peri-implantitis. Periodontal tissue biopsies (1 mmx I mmx I mm) from each patient were frozen in liquid nitrogen. Total RNA was extracted from these tissues, and the content, purity and integrity were detected. Specific primers were designed according to the sequence, and the mRNA expression levels of cellular fibronectin were detected by real-time PCR. The purity and integrity of the extracted total RNA were both high, and the specificity of amplified genes was very high with no other pollution. The mRNA expression of cFn in the adult periodontitis group (1.526+0.441) was lower than that in the healthy group (3.253+0.736). However, the mRNA expression of cFn in the peri-implantitis group (3.965+0.537) was significantly higher than that in the healthy group. The difference revealed that although both processes were destructive inflammatory reactions in the periodontium, the pathomechanisms were different and the variation started from the transcription level of the cFn gene.
文摘AIM: To design, optimize and validate a rapid,internally controlled real-time polymerase chain reaction(RT-PCR) test for herpes simplex virus(HSV) in the diagnosis of necrotizing herpes stromal keratitis.· METHODS: Tears alone or together with corneal epithelium scrapings from 30 patients(30 eyes)suspected of necrotizing herpes stromal keratitis were tested for HSV DNA by RT-PCR. The samples were collected during the first visit and then on the subsequent 7, 14, 28, 42, and 56 d. The symptoms of the patients were scored before treatment to determine the correlation between HSV concentration in the corneal epithelium scrapings and clinical scores.·RESULTS: The positive rate(46.4%) in the corneal epithelium group before the therapy was significantly higher than that(13.3%) in the tears group(P =0.006).There were 13 positive HSV patients before the therapy,the concentration of HSV DNA in corneal epithelium scrapings group was significantly higher than that in the tears group(paired t-test, P =0.0397). Multilevel mixedeffects model analysis showed that the difference between the corneal epithelium scrapings group and the tears group was statistically significant(P =0.0049). The Spearman rank correlation analysis indicated a positive correlation between the HSV concentration in the corneal epithelium scrapings and clinical scores before the treatment(r =0.844, P〈 0.0001).· CONCLUSION: RT-PCR appears to be a powerful molecular tool for the diagnosis of necrotizing herpes stromal keratitis.
文摘AIM: To develop a real-time reverse transcriptionpolymerase chain reaction(RT-PCR) assay to genotype rotavirus(G and P) in Alberta from January 2012 to June 2013. METHODS: We developed and validated a different approach to perform rotavirus G and P genotyping using a two-step SYBR green RT-PCR(rt-g PCR) by selecting genotype-specific primers of published conventional RT nested PCR(cn RT-PCR) assay and optimizing the amplification conditions. c DNA was first synthesized from total RNA with Super Script? Ⅱ reverse transcriptase kit followed by amplication step using monoplex SYBR green real-time PCR. After the PCR reaction, melting curve analysis was used to determine specific genotype. Sixteen samples previously genotyped using cn RT-PCR were tested using the new assay and the genotyping results were compared as sensitivity analysis. Assay specificity was evaluated by testing other gastroenteritis viruses with the new assay. The amplicon size of each available genotype was determined by gelelectrophoresis and DNA sequences were obtained using Sanger-sequencing method. After validation and optimization, the new assay was used to genotype 122 pediatric clinical stool samples previously tested positive for rotavirus using electron microscopy between January2012 and June 2013.RESULTS: The new rt-g PCR assay was validated and optimized. The assay detected G1 to G4, G9, G12 and P[4] and P[8] that were available as positive controls in our laboratory. A single and clear peak of melting curve was generated for each of specific G and P genotypes with a Tm ranging from 80 ℃ to 82 ℃. The sensitivity of rt-g PCR was comparable to cn RT-PCR with 100% correlation of the 16 samples with known G and P genotypes. No cross reaction was found with other gastroenteritis viruses. Using the new rt-g PCR assay, genotypes were obtained for 121 of the 122 pediatric clinical samples tested positive for rotavirus: G1P[8](42.6%), G2P[4](4.9%), G3P[8](10.7%), G9P[8](10.7%), G9P[4](6.6%), G12P[8](23.0%), and unknown GP[8](0.8%). For the first time, G12 rotavirus strains were found in Alberta and G12 was the second most common genotype during the study period. Gel electrophoresis of all the genotypes showed expected amplicon size for each genotype. The sequence data of the two G12 samples along with other genotypes were blasted in NCBI BLAST or analyzed with Rota C Genotyping tool(http://rotac.regatools.be/). All genotyping results were confirmed to be correct.CONCLUSION: rt-g PCR is a useful tool for the genotyping and characterization of rotavirus. Monitoring of rotavirus genotypes is important for the identification of emerging strains and ongoing evaluation of rotavirus vaccination programs.
基金Supported by grant from Fundamental Research Grant Scheme by Ministry of Higher Education(MoHE)600-IRMI/FRGS 5/3(101/2019).
文摘AIM:To investigate the stability of the seven housekeeping genes:beta-actin(ActB),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),18s ribosomal unit 5(18s),cyclophilin A(CycA),hypoxanthine-guanine phosphoribosyl transferase(HPRT),ribosomal protein large P0(36B4)and terminal uridylyl transferase 1(U6)in the diabetic retinal tissue of rat model.METHODS:The expression of these seven genes in rat retinal tissues was determined using real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR)in two groups;normal control rats and streptozotocininduced diabetic rats.The stability analysis of gene expression was investigated using geNorm,NormFinder,BestKeeper,and comparative delta-Ct(ΔCt)algorithms.RESULTS:The 36B4 gene was stably expressed in the retinal tissues of normal control animals;however,it was less stable in diabetic retinas.The 18s gene was expressed consistently in both normal control and diabetic rats’retinal tissue.That this gene was the best reference for data normalisation in RT-qPCR studies that used the retinal tissue of streptozotocin-induced diabetic rats.Furthermore,there was no ideal gene stably expressed for use in all experimental settings.CONCLUSION:Identifying relevant genes is a need for achieving RT-qPCR validity and reliability and must be appropriately achieved based on a specific experimental setting.
基金Grower/processor Check-off Funds administrated by theAmerican Sugar Cane League of the USA., Inc., Thibodaux, Louisiana, USA(to Y.-B. Pan)the Chinese 948 Project (2003-Q06) (to P.-H. Chen).
文摘Single pollen grain polymerase chain reaction (PCR) has succeeded in several species, however only limited numbers of pollen grains were involved due to difficulties in pollen isolation and lysis. This has limited its application in genetic analysis and mapping studies in plants. A high-throughput (HT) procedure for collecting and detecting genetic variation in a large number of individual pollen grains by PCR is reported. The HT procedure involved the collection of individual pollen grains by a pair of special forceps and the lysis of pollen grains in a heated alkali/detergent solution followed by neutralization with a tris-ethylenediamine tetraacetic acid (TE) buffer. These resulting template solutions yielded PCR reactions involving the 5S ribosomal RNA intergenic spacers, randomly amplified polymorphic DNA, and simple sequence repeats markers. Using this procedure, one person with experience could collect and process up to 288 single pollen grain PCR reactions per day. The method worked well on sugarcane, corn, Miscanthus spp., snap bean, sorghum, and tomato. The ability to collect and conduct PCR on individual pollen grains on a large scale offers a new approach to genetic analyses and mapping studies in an easily controllable environment with a considerable cost reduction. The method will also significantly benefit studies in species that are difficult subjects for classical genetic research.
基金supported by National Natural Science Foundation of China(Grant No.30671397 and No.30871655)the Public Beneficial Research Project of Agricultural Ministry,China(Grant No.nyhyzx07-056)
文摘Burkholderia glumae causing seedling rot and grain rot of rice was listed as a plant quarantine disease of China in 2007. It's quite necessary to set up effective detection methods for the pathogen to manage further dispersal of this disease. The present study combined the real-time PCR method with classical PCR to increase the detecting efficiency, and to develop an accurate, rapid and sensitive method to detect the pathogen in the seed quarantine for effective management of the disease. The results showed that all the tested strains of B. glumae produced about 139 bp specific fragments by the real-time PCR and the general PCR methods, while others showed negative PCR result. The bacteria could be detected at the concentrations of 1×10^4 CFU/mL by general PCR method and at the concentrations below 100 CFU/mL by real-time fluorescence PCR method. B. glumae could be detected when the inoculated and healthy seeds were mixed with a proportion of 1:100.
文摘Background The diagnosis of Pneumocystis pneumonia (PCP) in immunocompromised patients is still challenging today due to the absence of an in vitro culture system and the low diagnostic accuracy of microscopic examinations. Herein, we performed a meta-analysis to evaluate the accuracy of real-time polymerase chain reaction (PCR) in the diagnosis of PCP. Methods We searched Web of Knowledge and Medline from 1990 to May 2010 for studies reporting diagnostic accuracy data regarding the use of real-time PCR in the diagnosis of PCP in immunocompromised patients. Results Ten individual studies were included. Overall, the sensitivity of real-time PCR was 97% (95% CI: 93%-99%); the specificity was 94% (95% CI: 90%-96%). The area under the HSROC curve (95% CO for real-time PCR was 0.99 (0.97-0.99). In a subgroup analysis regarding studies involving HIV patients among the study population, the sensitivity and specificity were 97% (95% CI: 93%-99%) and 93% (95% CI: 89%-96%), respectively. Regarding studies using Bronchoalveolar lavage (BAL) samples only: sensitivity =98% (95% CI: 94%-99%); specificity =93% (95% CI: 89%- 96%), respectively. Regarding studies using microscopy as a reference standard: sensitivity =97% (95% CI: 92%-99%); specificity =93% (95% CI: 88%-96%). However, high between-study statistical heterogeneity was observed in all analyses. Conclusions Real-time PCR has a good diagnostic accuracy and may provide a useful adjunctive tool for the diagnosis of PCP in immunocompromised patients. Further studies are needed in order to identify any differences in the diagnostic performance of real-time PCR in HIV and non-HIV immunocompromised patients.
基金ACKNOWLEDGMENTS This research was supported by the Major State Basic Research Development Program of China (973 Program, Noso 2006GB504005 and 2009CB941700), the National Natural Science Foundation of China (No. 30872765) and the Basic Research Key Program of Shanghai (10]C1410800). Shi-Wei Duan is sponsored partly by the K. C. Wong Magna Fund of Ningbo University. Wethank Dr Ching-Ling Chen for kind suggestions regarding English in drafting this paper.
文摘Multiplex polymerase chain reaction (PCR) has been widely used to detect Y-chromosome micredeletions, which is one of the major causes of male infertility. Both the European Academy of Andrology (EAA) and the European Molecular Genetics Quality Network (EMQN) have recommended the use of sY84 and sY86 markers for the detection of azoospermia factor a (AZFa) microdeletion during DNA testing for male infertility. In this study, a large-scale analysis of AZF microdeletion in a total of 630 Chinese males, including healthy semen donors (n=200), infertile males with normal sperm count (n=226) and patients with either nonobstructive azoospermia or severe oligozoospermia (n=204), was performed. A series of nine sequence-tagged site (STS) markers from the AZF region of the Y chromosome was used to detect microdeletions. All primers were designed based on the recommendations of the National Center for Biotechnology Information. An unusually high incidence (73/630, 11.6%) of sY84-absent but sY86-present genotypes was observed in the AZFa microdeletion screening. Sequencing the sY84-flanking region revealed a total of 73 patients with sY84-absent but sY86-present genotypes have a T-to-G transversion at the fifth base from the 5' end of the reverse sY84 primer. These prevalent false positives, which were not only observed in infertile men, but also observed in donors, resulted from a single-nucleotide polymorphism (SNP) named rs72609647 in the targeting sequence of the reverse sY84 primer. Our study suggests that a pre-screening of existence of rs72609647 polymorphism can prevent the frequent false positive results of AZFa microdeletions detection in the infertile Chinese males. Given the SNP rs72609647 was recently found in a deep sequencing of a Chinese individual, the current EAA and EMQN standards may need to be scrutinized among different populations to avoid the potential genetic variations in the primer binding sequences.
基金Supported by 2010A310011 Henan Provincial Department of Education on Natural Science Research Projects
文摘AIM:To investigate the relationship between c.343A>G and c.2216A>C polymorphism sites in the CDH17 gene and colorectal carcinoma.METHODS:Ninety-three non-consanguineous colorectal carcinoma patients admitted to the Department of Oncology at the First Affiliated Hospital of Zhengzhou University were included in this study.Ninety-three peripheral venous blood samples,of approximately one milliliter from each patient,were collected betweenDecember 2009 and August 2010.The genomic DNA of these peripheral venous blood samples were extracted and purified using a Fermentas Genomic DNA Purification Kit(Fermentas,CA) according to the manufacturer' s protocol.The single nucleotide polymorphisms(SNPs) of the liver-intestine cadherin(CDH17) gene c.343A>G and c.2216A>C were determined by the polymerase chain reaction-single strand conformation polymorphism method(PCR-SSCP) in 93 peripheral venous blood samples from patients suffering with colorectal carcinoma.Typical samples that showed different migration bands in SSCP were confirmed by sequencing.Directed DNA sequencing was used to check the correctness of the genotype results from the PCR-SSCP method.RESULTS:There was a significant association between the c.2216 A>C SNPs of the CDH17 gene and the tumor-node-metastasis(TNM) grade,as well as with lymph node status,in 93 peripheral venous blood samples from colorectal carcinoma patients.The genotype frequencies of A/C,A/A,and C/C were 12.90%,33.33% and 53.76%,respectively.There was a significant correlation between lymph node metastasis,TNM grade,and the genotype distribution(P < 0.05).The C/C genotype raised the risk of lymph node metastasis and the TNM grade.There was a significant difference in the TNM grade and lymph node metastasis between the A/A and C/C genotypes(P = 0.003 and P = 0.013,respectively).Patients with colorectal carcinoma carrying the C allele tended to have a higher risk of lymph node metastasis and have a higher TNM grade.The difference between the TNM grades,as well as the lymph node metastasis of the two alleles,was statistically significant(P < 0.01).CONCLUSION:The SNPs of the CDH17 gene c.2216 A>C might be clinically important in the prognosis of colorectal carcinoma.
文摘The rRNA genetic locus is found in all prokaryotic organisms, and is highly conservative, although its relatively stable variations are found frequently in different bacteria. The utility of this locus as a taxonomic and phylogenetic tool has been reported widely. This study, aimed at 16S rRNA gene (16S rDNA) and with the help of biomolecular methods, attempted to achieve the goal of rapid identification of common pathogens. In this study, 333 clinical isolated pathogenic bacteria were collected. Two pairs of primers were chosen and labeled with different fluorescent dyes and then used to amplify the genomic DNA extracted from bacteria. The PCR products were then detected by capillary electrophoresis-single strand conformation polymorphism (CE-SSCP). In order to pursue higher resolution and peak-separation effect, a high efficient separating medium, liner polyacrylamidedel (LPA), was put to use in this study. Finally, every bacteria colony generated distinct patterns from each other, which were easily to be used for identification. These results indicated that PCR-CE-SSCP was a rapid identification method for bacterial identification, with the aspects of high efficiency and high precision. Compared with traditional method, this technology is of great utility for clinical use especially for its high sensitivity.
基金This project was supported by a grant from Science Foun-dation of Ministry of Public Heath of China (No. 96 .2 - 112 )and a grant from Hubei Provincial National Natural ScienceFoundation(96 J0 6 8)
文摘The single cell isolation technique was used to detect fetal nucleated erythroblasts at a single cell level from the peripheral blood of pregnant women in order to investigate the feasibility of this method for noninvasive prenatal diagnosis. Single fetal nucleated erythroblasts were isolated from the peripheral blood samples from 51 pregnant women by micromanipulation techniques after density gradient centrifugation. Nested polymerase chain reaction method was used to amplify the SRY gene. It was found that the concordance rate of amplification results with real fetal sex was 82.61 %. The sensitivity and specificity were 80 % and 87.50 % respectively. It was suggested that it is feasible and promising in non invasive prenatal diagnosis to detect fetal nucleated erythroblasts at a single cell level by using micromanipulation techniques.
文摘The genomic DNA is the organism’s various physiological and pathological characteristics of the material base.Single nucleotide polymorphisms(single nucleotide polymorphisms,SNPs) is the genome of the most common form of genetic variation in the human genome widespread.With the completion of the Human Genome Project to stimulate,there is growing focus on research developed in this latest 3rd generation of genetic markers of practical significance.This paper describes the classification of SNPs and characteristics of the human genome SNP s research and development and the present in today’s practice,the application of design experimts to explore the direct sequencing of amplified using PCR-based method showed that SNPs.
文摘BACKGROUND Head and neck squamous cell carcinoma(HNSCC) is considered to be a progressive disease resulting from alterations in multiple genes regulating cell proliferation and differentiation like receptor tyrosine kinases(RTKs) and members of the fibroblast growth factor receptors(FGFR)-family. Singlenucleotide polymorphism(SNP) Arg388 of the FGFR4 is associated with a reduced overall survival in patients with cancers of various types. We speculate that FGFR4 expression and SNP is associated with worse survival in patients with HSNCC.AIM To investigate the potential clinical significance of FGFR4 Arg388 in the context of tumors arising in HNSCC, a comprehensive analysis of FGFR4 receptor expression and genotype in tumor tissues and correlated results with patients' clinical data in a large cohort of patients with HNSCC was conducted.METHODS Surgical specimens from 284 patients with HNSCC were retrieved from the Institute of Pathology at the Ludwig-Maximilian-University in Germany.Specimens were analyzed using immunohistochemistry and polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP). The expression of FGFR4 was analyzed in 284 surgical specimens of HNSCC using immunohistochemstry. FGFR4 polymorphism was detected by PCR-RFLP.Patients' clinical data with a minimum follow-up of 5 years were statistically evaluated with a special emphasis on survival analysis employing Kaplan-Meier estimator and Cox regression analysis.RESULTS Concerning the invasive tumor areas the intensity of the FGFR4 expression was evaluated in a four-grade system: no expression, low expression, intermediate and high expression. FGFR4 expression was scored as "high"(+++) in 74(26%),"intermediate"(++) in 103(36.3%), and "low"(+) in 107(36.7%) cases. Analyzing the FGFR4 mutation it was found in 96 tumors(33.8%), 84 of them(29.6%) having a heterozygous and 12(4.2%) homozygous mutated Arg388 allele. The overall frequency concerning the mutant alleles demonstrated 65% vs 34% mutated alleles in general. FGFR4 Arg388 was significantly associated with advanced tumor stage(P < 0.004), local metastasis(P < 0.0001) and reduced disease-free survival(P < 0.01). Furthermore, increased expression of FGFR4 correlated significantly with worse overall survival(P < 0.003).CONCLUSION In conclusion, the FGFR4 Arg388 genotype and protein expression of FGFR4 impacts tumor progression in patients with HNSCC and may present a useful target within a multimodal therapeutic intervention.
文摘Aim To use and modify the molecular histology technique that was first introduced by Hansmann et al and to investigate the cell origin and the clonality of Hodgkin/Reed Sternberg (H/R S) cell. Method Single H/R S cells were isolated by micromanipulation from frozen histological sections of tissues affected by Hodgkins disease. After DNA was extracted from these cells, PCR was performed with primers designed for β globin gene that exists in any somatic cells. Results The results showed that a special PCR product (268bp) was found in 12 out of 38 H/R S cells (positive rate of 31.6% ) with DNA from the cells micropicked from the frozen sections, whereas no any products was found with the DNA from these cells of formalin fixed and paraffin embedded sections. Conclusions The molecular histology technique we used is suitable for detecting the immunoglobulin heavy and light chain gene rearrangement in single H/R S cells.
基金Supported by the Key Program of Science and Technology Innovation in Ningbo (No.2019B10009)the National Natural Science Foundation of China (No.41476111)。
文摘The quantitative realtime polymerase chain reaction(RT-qPCR)is a powerful and sensitive method to measure expression of targeted gene but it highly relies on the use of suitable reference genes for data normalization.We evaluated the expressions of 8 housekeeping genes:18S ribosomal rDNA(18S rDNA),28S ribosomal r DNA(28S rDNA),rubisco large subunit(rbc-L),β-actin(ACT),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),elongation factor 1(EF1),β-tubulin(Tub B),and P-phycoerythrin B(PEB),to select the suitable reference genes for different life-history stages(tetrasporophyte,carposporophyte,and male/female gametophyte)of Gracilaria vermiculophylla by absolute quantitative method.Softwares geNorm and BestKeeper were used to verify the results acquired from copy number analysis.Results show that the expression of identified reference genes varied in comparing groups composed of different type of life stages.It is suggested that 18S rDNA and TubB could be used for highly complex samples composed of mixed ploidy and phases.18S rDNA and 28S rDNA were also preferred for using among the matured isomorphic samples.But for samples with different maturities,TubB and ACT were recommended for tetrasporophytes and gametophytes respectively.