BACKGROUND Hepatitis C virus(HCV),hepatitis B virus(HBV),and human immunodeficiency virus 1(HIV-1)are the most epidemic blood-borne viruses,posing threats to human health and causing economic losses to nations for com...BACKGROUND Hepatitis C virus(HCV),hepatitis B virus(HBV),and human immunodeficiency virus 1(HIV-1)are the most epidemic blood-borne viruses,posing threats to human health and causing economic losses to nations for combating the infection transmission.The diagnostic methodologies that depend on the detection of viral nucleic acids are much more expensive,but they are more accurate than sero-logical testing.AIM To develop a rapid,cost-effective,and accurate diagnostic multiplex polymerase chain reaction(PCR)assay for simultaneous detection of HCV,HBV,and HIV-1.METHODS The design of the proposed PCR assay targets the amplification of a short conserved region featured with a distinguishable melting profile and electro-phoretic molecular weight inside each viral genome.Therefore,this diagnostic method will be appropriate for application in both conventional(combined with electrophoresis)and real-time PCR facilities.Confirmatory in silico investigations were conducted to prove the capability of the approached PCR assay to detect variants of each virus.Then,Egyptian isolates of each virus were subjected to the wet lab examination using the given diagnostic assay.RESULTS The in silico investigations confirmed that the PCR primers can match many viral variants in a multiplex PCR assay.The wet lab experiment proved the efficiency of the assay in distinguishing each viral type through high-resolution melting analysis.Compared to related published assays,the proposed assay in the current study is more sensitive and competitive with many expensive PCR assays.CONCLUSION This study provides a simple,cost-effective,and sensitive diagnostic PCR assay facilitating the detection of the most epidemic blood-borne viruses;this makes the proposed assay promising to be substitutive for the mistakable and cheap serological-based assays.展开更多
AIM: To study persistence and replication of hepatitis C virus (HCV) in patients' peripheral blood mononuclear cells (PBMC) cultured in vitro. METHODS: Epstein Barr virus (EBV) was used to transform the hepatitis ...AIM: To study persistence and replication of hepatitis C virus (HCV) in patients' peripheral blood mononuclear cells (PBMC) cultured in vitro. METHODS: Epstein Barr virus (EBV) was used to transform the hepatitis C virus from a HCV positive patient to permanent lymphoblastoid cell lines (LCL). Positive and negative HCV RNA strands of the cultured cells and growth media were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) each month. Core and NS5 proteins of HCV were further tested using immunohistochemical SP method and in situ RT-PCR. RESULTS: HCV RNA positive strands were consistently detected the cultured cells for one year. The negative-strand RNA in LCL cells and the positive-strand RNA in supernatants were observed intermittently. Immunohistochemical results medicated expression of HCV NS3 and C proteins in LCL cytoplasm mostly. The positive signal of PCR product was dark blue and mainly localized to the LCL cytoplasm. The RT-PCR signal was eliminated by overnight RNase digestion but not DNase digestion. CONCLUSION: HCV may exist and remain functional in a cultured cell line for a long period.展开更多
Objective: To detect circulating hepatocellular carcino-ma by demonstrating hepatocellular carcinoma cells orhepatocyte-associated mRNA in the nuclear cell com-ponent of peripheral blood (PBL).Methods: Peripheral bloo...Objective: To detect circulating hepatocellular carcino-ma by demonstrating hepatocellular carcinoma cells orhepatocyte-associated mRNA in the nuclear cell com-ponent of peripheral blood (PBL).Methods: Peripheral blood (5 ml) samples were ob-tained from 93 patients with hepatocellular carcinoma(HCC) and from 33 control subjects (9 with liver cir-rhosis after hepatitis B,14 with chronic hepatitis B,10with normal liver function). To identify HCC cells inperipheral blood, liver-specific human alpha-fetopro-tein (AFP) mRNA was amplified from total RNA ex-tracted from whole blood by reverse transcription-polymerase chain reaction.Results: AFPmRNA was detected in 50 blood samplesfrom the HCC patients (50/93, 53.8%). In contrast,there were no clinical control patients whose samplesshowed detectable AFPmRNA in PBL. The presence ofAFPmRNA in blood seemed to be correlated with thestage (by TNM classification) of HCC, the serum AFPvalue, and the presence of intrahepatic metastasis,portal vein thrombosis, tumor diameter and/or distantmetastasis. In addition, AFPmRNA was detected in theblood of 21 patients with metastasis at extrahepaticorgans (100%) in contrast to 29 (40.3%)of 72 pa-tients without metastasis.Conclusion: The presence of AFPmRNA in peripheralblood may be an indicator of malignant hepatocytes,which might predict hematogenous spreading metasta-sis of tumor cells in patients with HCC.展开更多
An easy and reliable method was developed for construction and quantification of competitive templates, which shared the same sequence as the amplified target DNA except for a 20 bp insertion in the middle by recombi...An easy and reliable method was developed for construction and quantification of competitive templates, which shared the same sequence as the amplified target DNA except for a 20 bp insertion in the middle by recombinant polymerase chain reaction (PCR). Among the advantages of competitive PCR is that any predictable or unpredictable variable that affects amplification has the same effect on both target and competitor species and that the final ratio of amplified products reflects exactly the initial targets. The utilization of a thermostable reverse transcriptase in the RT step was proposed to overcome the problem of the efficiency of target cDNA synthesis. In addition, to obtain reliable measurements, it was recommended to perform four PCR with amounts of competitive template flanking the concentration of the target mRNA.展开更多
Quantitative real-time reverse transcription-polymerase chain reaction （qPCR） is widely used to investigate transcriptional changes following experimental manipulations to the nervous system. Despite the widespread ...Quantitative real-time reverse transcription-polymerase chain reaction （qPCR） is widely used to investigate transcriptional changes following experimental manipulations to the nervous system. Despite the widespread utilization of qPCR, the interpretation of results is marred by the lack of a suitable reference gene due to the dynamic nature of endogenous transcription. To address this inherent deficiency, we investigated the use of an exogenous spike-in mRNA, luciferase, as an internal reference gene for the 2ct normalization method. To induce dynamic transcription, we systemically administered capsaicin, a neurotoxJn selective for C-type sensory neurons expressing the TRPV-1 receptor, to adult male Sprague-Dawley rats. We later isolated nodose ganglia for qPCR analysis with the reference being either exogenous luciferase mRNA or the commonly used endogenous reference 13-111 tubulin. The exogenous luciferase mRNA reference clearly demonstrated the dynamic expression of the endogenous reference. Furthermore, variability of the endogenous reference would lead to misinterpretation of other genes of interest. In conclusion, traditional reference genes are often unstable under physiologically normal situations, and certainly unstable following the damage to the nervous system. The use of exogenous spike-in reference provides a consistent and easily implemented alternative for the analysis of qPCR data.展开更多
In order to detect circulating cells of hepatocellular carcinoma(HCC) in the peripheral blood with reverse transcripition polymerase chain reaction (RT-PCR ), alpha-fetoprotein (AFP ) mRNA was tested in the blood samp...In order to detect circulating cells of hepatocellular carcinoma(HCC) in the peripheral blood with reverse transcripition polymerase chain reaction (RT-PCR ), alpha-fetoprotein (AFP ) mRNA was tested in the blood samples of 113 cases of HCC and 69 controls (including 30 cases of liver cirrhosis, 9 cases of metastatic liver cancer and 30 normal subjects). 20/43 (46. 5% ) cases of HCC and 2/30 (6. 7% ) cases of liver cirrhosis are positive and the cases of nletastatic liver cancer and normal controls were negative for human AFP(hAFP) rnRNA. The presence of hAFP mRNA in the peripheral blood seems to be correlated with intrahepatic and distant nletastasls of HCC and portal vein thrombosis. It is concluded that the presence of hAFP mRNA in the peripheral hloocl is an indicator of circulating HCC cells and can be used to diagnose the rnetastasisof HCC through henlatogenous route and RT-PCR amplification of hAFP mRNA is a sensitive and specificprocedure for detecting circulating cells of HCC.展开更多
Objective： To explore an ideal approach for detecting the physical status of HPV-16 in clinic use and to investigate the integrated HPV-16 in CINs and cervical cancer. Methods： Multiplex real-time PCR method was est...Objective： To explore an ideal approach for detecting the physical status of HPV-16 in clinic use and to investigate the integrated HPV-16 in CINs and cervical cancer. Methods： Multiplex real-time PCR method was established to quantify the copy numbers of E2 and E6 genes （E2/E6） for analysis of the physical status of HPV-16 DNA and this assay was compared to Southern blot analysis. HPV-16-containing paraffin-embedded tissues including 49 CINs and 51 cervical squamous cancers were detected using the method. Results： （1） The cutoff ratio of E2/E6 to distinguish pure episomal from mixed HPV-16, was 0.81 in the multiplex real-time PCR; （2） The agreement rate between multiplex real-time PCR and Southern blot was 81.5% （the Kappa statistic was 0.844, P〈0.001）; （3） HPV-16 DNA existed in an episomal form in 57.1% and mixed form in 42.9% of CIN I lesions; The concomitant form of HPV-16 （〉70%） constituted the majodty in CIN Ⅱ and CIN Ⅲ; HPV-16 DNA mostly integrated into the host chromosome （s） in squamous cervical cancers （68.6%）; （4） The incidence of HPV-16 integration was increased with the degree of cervical lesions; （5） The frequency of pure integrated HPV-16 in stage Ⅱ＋Ⅲ （88%） was significantly higher than that in stage Ⅰ （33.3%）. Conclusion： （1） Mutiplex real-time PCR provides a rapid, sensitive and reliable method for clinic detection of the physical state of HPV-16 DNA; （2） The integration of the HPV-16 DNA is a very eady and important event in the progression from preinvasive to invasive cervical cancer; （3） The pure integrated status of HPV-16 in cervical cancer may be associated with poor prognosis of cervical cancer, but further study will be needed to prove its prognostic significance.展开更多
Objective To establish a multiplex polymerase chain reaction （M-PCR） assay for simultaneous detection of pathogens causing genital ulcer disease （GUD）. Mothods Based on the gene-specific region of the following p...Objective To establish a multiplex polymerase chain reaction （M-PCR） assay for simultaneous detection of pathogens causing genital ulcer disease （GUD）. Mothods Based on the gene-specific region of the following pathogens： Chlamydia trachomatis omp l/ompb, herpes simplex virus （HSV） DNA polymerase, Treponema pollidum tpp47, Haemophilus ducreyi 16s rRNA, four sets of primers were designed and an M-PCR assay was developed to detect four pathogens in one test. The assay was evaluated with diagnostic result of golden standard for each pathogen.Results Of the 51 clinical samples, M-PCR showed slightly higher positive rate （47.1%） of HSV than cell culture （23.6%）. Meanwhile, the positive rate of T. pallidum detected by M-PCR and dark-field microscopy was 19.6% （10/51） and 15.7% （8/51）, respectively. Only one sample was positive for H. ducreyi and no sample was positive for C. trachomatis detected by both M-PCR assay and culture. Conclusion This primary study indicated that M-PCR assay can simultaneously and rapidly detect the four etiologic pathogens causing GUD.展开更多
BACKGROUND Recently,stool multiplex polymerase chain reaction(PCR)tests have been developed for identifying diarrhea-causing bacterial pathogens.Furthermore,fecal calprotectin is a well-known effective marker for inte...BACKGROUND Recently,stool multiplex polymerase chain reaction(PCR)tests have been developed for identifying diarrhea-causing bacterial pathogens.Furthermore,fecal calprotectin is a well-known effective marker for intestinal mucosal inflammation.AIM To evaluate the efficacy of stool multiplex PCR and fecal calprotectin in acute infectious diarrhea.METHODS Overall,400 patients with acute infectious diarrhea were enrolled from Kangdong Sacred Heart Hospital(January 2016 to December 2018).Multiplex PCR detected 7 enteropathogenic bacteria including Salmonella,Campylobacter,Shigella,Escherichia coli O157:H7,Aeromonas,Vibrio,and Clostridium difficile.We reviewed clinical and laboratory findings using stool multiplex PCR.RESULTS Stool multiplex PCR test detected considerably more bacterial pathogens than stool culture(49.2%vs 5.2%),with Campylobacter as the most common pathogen(54%).Patients with positive stool PCR showed elevated fecal calprotectin expression compared to patients with negative stool PCR(1124.5±816.9 mg/kg vs 609±713.2 mg/kg,P=0.001).C-reactive protein(OR=1.01,95%CI:1.001-1.027,P=0.034)and sigmoidoscopy-detected colitis(OR=4.76,95%CI:1.101-20.551,P=0.037)were independent factors in stool PCR-based detection of bacterial pathogens.Sensitivity and specificity of calprotectin were evaluated to be 70.5%and 60.9%,respectively(adjusted cut-off value=388 mg/kg).CONCLUSION Stool multiplex PCR test has increased sensitivity in detecting pathogens than conventional culture,and it is correlated with calprotectin expression.Stool multiplex PCR and calprotectin may be effective in predicting clinical severity of infectious diarrhea.展开更多
OBJECTIVE: To assess the feasibility of detecting SYT-SSX fusion transcripts in paraffin-embedded tissues of synovial sarcoma by reverse transcription-polymerase chain reaction (RT-PCR). METHODS: RT-PCR was used to am...OBJECTIVE: To assess the feasibility of detecting SYT-SSX fusion transcripts in paraffin-embedded tissues of synovial sarcoma by reverse transcription-polymerase chain reaction (RT-PCR). METHODS: RT-PCR was used to amplify the SYT-SSX fusion transcripts using archival formalin-fixed paraffin-embedded tumor specimens from a series of 37 synovial sarcoma cases. To investigate the specificity of the SYT-SSX fusion transcripts, a variety of non-synovial sarcoma tumors were included in the study as negative controls. The detected messages derived from fusion genes were confirmed by subsequent sequence analysis. RESULTS: SYT-SSX fusion transcripts were detected in 33 of 37 (89.2%) synovial sarcomas. None of the 34 cases of non-synovial sarcoma tumors showed amplified products of SYT-SSX fusion transcripts, although PBGD mRNA was detected in all specimens. Among 33 SYT-SSX-positive synovial sarcomas, 22 tumors had an SYT-SSX 1 fusion transcript, whereas 6 tumors had an SYT-SSX2 fusion transcript. Fusion types can not be distinguished in the remaining 5 cases. There was a significant relationship between SYT-SSX fusion type and histologic subtype. All 10 biphasic synovial sarcomas had the SYT-SSX1 fusion, whereas all tumors with SYT-SSX2 were of monophasic morphology (P展开更多
Objective: To access the performance of the Tellgenplex human papillomavirus(HPV) DNA test compared to the polymerase chain reaction-reverse dot blot(PCR-RDB) assay for the HPV genotyping.Methods: Sixty cervical swab ...Objective: To access the performance of the Tellgenplex human papillomavirus(HPV) DNA test compared to the polymerase chain reaction-reverse dot blot(PCR-RDB) assay for the HPV genotyping.Methods: Sixty cervical swab samples were genotyped by the Tellgenplex HPV DNA test and the PCR-RDB assay.The Tellgenplex HPV DNA test and the PCR-RDB assay can detect 26 and 23 HPV genotypes, respectively.Each sample showed discrepancy was genotyped using sequencing.Results: The percent agreement between the two tests ranged from 83.3% to 100.0% according to different genotype.This showed perfect agreement(>0.81) for high-risk HPV genotypes(35, 39, 45, 53, 56, 59, 66, 68, and 82), substantial agreement(>0.65) for high-risk HPV genotypes(16, 18, 33, 52, and 58) and low-risk HPV genotype 43 between the two assays by the kappa analysis.The positive rates of the two assays for frequent HPV genotypes(16, 35, 39, 45, 52, 53, 58, 59, 66, and 82) were not statistically different, but the PCR-RDB assay showed higher positive rates than the Tellgenplex HPV DNA test for HPV genotypes 81(P<0.05).As for more than 10 positive results by the Tellgenplex HPV DNA test and/or the PCR-RDB assay, the PCR-RDB assay showed higher relative sensitivity and specificity than the Tellgenplex HPV DNA test for the three HPV genotypes(16, 52, and 81).All HPV genotypes that can be detected by only the Tellgenplex HPV DNA test(HPV genotypes 44 and 55) were confirmed by sequencing.Conclusions: In conclusion, our results demonstrated that the PCR-RDB assay which can detect more multiple HPV genotypes in each specimen shows higher relative sensitivity and specificity than the Tellgenplex HPV DNA test, which makes it a better option for routine clinical use.展开更多
A set of allele-specific oligonucleotide (ASO) probes used for detecting all 18 β-tha-lassemia mutations found in Chinese was immobilized on two strips of Biodyne C membrane;one containing 7 pairs of oligonucleotide ...A set of allele-specific oligonucleotide (ASO) probes used for detecting all 18 β-tha-lassemia mutations found in Chinese was immobilized on two strips of Biodyne C membrane;one containing 7 pairs of oligonucleotide probes specific for the most commonly found mutant al-leles,and the other containing the remaining 11 pairs of ASO_s specific for the less commonlyfound.The membranes were hybridized with β-globin sequences amplified by polymerase chainreaction (PCR) with biotinylated primers,and then treated with Streptavidin-POD conjugateand substrates for color development.The method has been applied successfully to the detectionof all 18 Chinese β-thalassemia mutations and prenatal diagnosis of two high-risk pregnancies ofβ-thalassemia.Patients with homozygous,heterozygous and compound heterozygous alleles ofthese mutations and normal individuals could be easily distinguished by the present method.Us-ing the immobilized-probe format (reverse dot blot),it was able to screen simultaneously multi-ple β-thalassemia mutations of a DNA sample by performing hybridization only once.This assayis simple,rapid and independent of radio-isotopes and can be appplied for all 18 β-thalassemiamutations so far found in Chinese population.It is considered that this method may be usefulfor gene frequency investigation of large numbers of β-thalassemia DNA samples and used as aroutine method in the clinic laboratory.展开更多
SRY (sex-determining region Y chromosome) is considered as a strong candidste for the TDF (testis determining factor) and has been cloned following another candidate ZFY (zinc finger protein gene). In this study,eight...SRY (sex-determining region Y chromosome) is considered as a strong candidste for the TDF (testis determining factor) and has been cloned following another candidate ZFY (zinc finger protein gene). In this study,eight cases of sex revelsal, including four 46, XX males and four 46, XY females aere examined for the presence of SRY sequence and a Y-repeated DNA locus. Our data indicated that the genownic DNA of the four classical 46,XX males had the SRY sequences. On the other hand, both SRY sequences and Ye repeated DNA sequences were present in all four 46, XY females.These resultssuavest that SAY sequences were responsible for the sexraversal of 46, XX males Whereas there may be other genetic mechanisms forthe sex reversal of 46, XY females Without the lack of SRY sequences.展开更多
GBV C/HGV is a newly identified virus associated with human hepatitis In this study, the nucleotide sequences of the partial NS5 gene of GBV C/HGV derived from sera of 8 Chinese patien...GBV C/HGV is a newly identified virus associated with human hepatitis In this study, the nucleotide sequences of the partial NS5 gene of GBV C/HGV derived from sera of 8 Chinese patients were determined The nucleotide homology among the 8 isolates were 92% on average On the basis of sequence analysis, two sets of oligonucleotide primers derived from highly conserved region of GBV C/HGV NS5 gene were designed to establish both sensitive and specific nested PCR for detection of GBV C/HGV RNA 253 Chinese patients were examined for the virus RNA GBV C/HGV RNA positive rates in patients infected with HBV, HCV and patients with chronic non B,non C hepatitis were 18 4%, 19 8% and 8 9% respectively This result suggested that HBV,HCV and GBV C/HGV shared the same transmission risk factors 8 patients with GBV C/HGV and HCV coinfection were retrospectively observed for the response to interferon Coinfection with GBV/HGV did not negatively influence the responsiveness of HCV, and GBV C/HGV was sensitive to interferon to a certain degree展开更多
A reproducible,quantitative,non-radioactive method for the analysis of mRNA expression is described.After RNA preparation and cDNA synthesis,the cDNA was co-amplified with an internal standard in the same PCR system.T...A reproducible,quantitative,non-radioactive method for the analysis of mRNA expression is described.After RNA preparation and cDNA synthesis,the cDNA was co-amplified with an internal standard in the same PCR system.The PCR products containing both targen and internal standard amplificates were electrophoresed and detected on an ABI 377 DNA Sequencer.For each sample,β-actin was also quantified by an identical procedure to compensate for relative differences between samples in the integrity of the individual RNA samples and for variations in reverse transcription.Due to the linear relationship between cDNA content and PCR product ratio of target cDNA template and competitive standard,a single PCR reaction was sufficient for quantification of a sample.The experimental results showed that the method is a mRNA quantitative RT-PCR method with high sensitivity and good reproducibility.It can be used in large-scale accurate quantitative analyses of mRNA expression of any gene.展开更多
Background:Patients carrying the HongKongαα(HKαα)allele and-α3.7/αααanti-4.2 could be misdiagnosed as-α3.7/ααby the current conventional thalassemia detection methods,leading to inaccurate genetic counselin...Background:Patients carrying the HongKongαα(HKαα)allele and-α3.7/αααanti-4.2 could be misdiagnosed as-α3.7/ααby the current conventional thalassemia detection methods,leading to inaccurate genetic counseling and an incorrect prenatal diagnosis.This study was aimed to accurately analyze the genotypes of HKααcarriers and-α3.7/αααanti-4.2.Methods::Samples were collected in our hospital from July 2017 to October 2019.Twenty-four common types of Chinese thalassemia were screened by gap-polymerase chain reaction(Gap-PCR)and reverse dot blot(RDB).Anti-4.2 multiplex-PCR was used to confirm carriers of theαααanti-4.2 duplication with-α3.7 deletion.Two-round nested PCR and multiplex ligation-dependent probe amplification(MLPA)were applied to accurately identify and confirm their genotypes.For data analysis,we used descriptive statistics and Fisher’s exact tests.Results::Two thousand five hundred and forty-four cases were identified as thalassemia in 5488 peripheral blood samples.The results showed thatα,β,andαβcompound thalassemia were identified in 1190(46.78%),1286(50.55%),and 68(2.67%)cases,respectively.A total of 227 samples from thalassemia patients were identified as-α3.7/ααby Gap-PCR,and the genotypes of two samples were uncertain.There was a difference between Gap-PCR and combined groups(Gap-PCR combined with nested PCR and MLPA)in detecting HKαα(P<0.05).Among the 229 patients,20 patients were identified as HKααcarriers and one was identified as-α3.7/ααα anti-4.2 by two-round nested PCR and MLPA,including 15 patients with HKαα/αα,three with HKαα/αα and β-thalassemia coinheritance,one with HKαα/-SEA,one with HKαα/-α4.2 andβ-thalassemia coinheritance,and one with-α3.7/αααanti-4.2 and β-thalassemia coinheritance.Conclusions::αααanti-4.2 and HKααgenotypes of patients carrying-α3.7 need to be detected to reduce the misdiagnosis rate of patients carrying HKααand-α3.7/αααanti-4.2 alleles.More accurate genetic counseling can be provided in the clinic using nested PCR combined with MLPA.展开更多
Trisomy 21, also named Down syndrome was the most frequent autosomal aneuploidy and the most common cause of mental retardation. Fifty percent patients had congenital heart malformation. Every 20 minutes one case of t...Trisomy 21, also named Down syndrome was the most frequent autosomal aneuploidy and the most common cause of mental retardation. Fifty percent patients had congenital heart malformation. Every 20 minutes one case of trisomy 21 was born, and the incidence rate was 1 in 600 to 800 newborns in China.1 In two thirds of cases with trisomy 21, there was a spontaneous abortion, so the actual incidence was higher than that obtained postnatally.展开更多
Among different endangered animal species,snakes are the most neglected creature looked at with apathy and therefore,are ruthlessly killed,illegally trafficked,and poached for their venom,lucrative skin,meat,and bones...Among different endangered animal species,snakes are the most neglected creature looked at with apathy and therefore,are ruthlessly killed,illegally trafficked,and poached for their venom,lucrative skin,meat,and bones for manufacturing of medicines,accessories,and food items.Establishing the identity of the endangered snake species is important for punishing the offenders under Wildlife Protection Act(WPA)(1972)but morphological characters fail to establish identity as they are often altered.The technique of identification of snake species at molecular level holds very effective conclusion in punishing offender.Here,we have constructed and demonstrated a novel multiplexing polymerase chain reaction technique,using 16S rRNA and C-mos gene for identification of four Indian snake species,namely Ptyas mucosa,Daboia russellii,Naja naja,and Xenochrophis piscator.They are listed in Appendix-II and III of convention on international trade in endangered species of wild fauna and flora and Schedule II;Part II of Indian WPA,1972.Therefore,it may be considered a functional tool for establishing species-specific identity of four Indian snake species and promising to be useful for their conservation.展开更多
文摘BACKGROUND Hepatitis C virus(HCV),hepatitis B virus(HBV),and human immunodeficiency virus 1(HIV-1)are the most epidemic blood-borne viruses,posing threats to human health and causing economic losses to nations for combating the infection transmission.The diagnostic methodologies that depend on the detection of viral nucleic acids are much more expensive,but they are more accurate than sero-logical testing.AIM To develop a rapid,cost-effective,and accurate diagnostic multiplex polymerase chain reaction(PCR)assay for simultaneous detection of HCV,HBV,and HIV-1.METHODS The design of the proposed PCR assay targets the amplification of a short conserved region featured with a distinguishable melting profile and electro-phoretic molecular weight inside each viral genome.Therefore,this diagnostic method will be appropriate for application in both conventional(combined with electrophoresis)and real-time PCR facilities.Confirmatory in silico investigations were conducted to prove the capability of the approached PCR assay to detect variants of each virus.Then,Egyptian isolates of each virus were subjected to the wet lab examination using the given diagnostic assay.RESULTS The in silico investigations confirmed that the PCR primers can match many viral variants in a multiplex PCR assay.The wet lab experiment proved the efficiency of the assay in distinguishing each viral type through high-resolution melting analysis.Compared to related published assays,the proposed assay in the current study is more sensitive and competitive with many expensive PCR assays.CONCLUSION This study provides a simple,cost-effective,and sensitive diagnostic PCR assay facilitating the detection of the most epidemic blood-borne viruses;this makes the proposed assay promising to be substitutive for the mistakable and cheap serological-based assays.
基金The paper was support by a grant from the Ministry Youth Research of China,No.98-1-269
文摘AIM: To study persistence and replication of hepatitis C virus (HCV) in patients' peripheral blood mononuclear cells (PBMC) cultured in vitro. METHODS: Epstein Barr virus (EBV) was used to transform the hepatitis C virus from a HCV positive patient to permanent lymphoblastoid cell lines (LCL). Positive and negative HCV RNA strands of the cultured cells and growth media were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) each month. Core and NS5 proteins of HCV were further tested using immunohistochemical SP method and in situ RT-PCR. RESULTS: HCV RNA positive strands were consistently detected the cultured cells for one year. The negative-strand RNA in LCL cells and the positive-strand RNA in supernatants were observed intermittently. Immunohistochemical results medicated expression of HCV NS3 and C proteins in LCL cytoplasm mostly. The positive signal of PCR product was dark blue and mainly localized to the LCL cytoplasm. The RT-PCR signal was eliminated by overnight RNase digestion but not DNase digestion. CONCLUSION: HCV may exist and remain functional in a cultured cell line for a long period.
文摘Objective: To detect circulating hepatocellular carcino-ma by demonstrating hepatocellular carcinoma cells orhepatocyte-associated mRNA in the nuclear cell com-ponent of peripheral blood (PBL).Methods: Peripheral blood (5 ml) samples were ob-tained from 93 patients with hepatocellular carcinoma(HCC) and from 33 control subjects (9 with liver cir-rhosis after hepatitis B,14 with chronic hepatitis B,10with normal liver function). To identify HCC cells inperipheral blood, liver-specific human alpha-fetopro-tein (AFP) mRNA was amplified from total RNA ex-tracted from whole blood by reverse transcription-polymerase chain reaction.Results: AFPmRNA was detected in 50 blood samplesfrom the HCC patients (50/93, 53.8%). In contrast,there were no clinical control patients whose samplesshowed detectable AFPmRNA in PBL. The presence ofAFPmRNA in blood seemed to be correlated with thestage (by TNM classification) of HCC, the serum AFPvalue, and the presence of intrahepatic metastasis,portal vein thrombosis, tumor diameter and/or distantmetastasis. In addition, AFPmRNA was detected in theblood of 21 patients with metastasis at extrahepaticorgans (100%) in contrast to 29 (40.3%)of 72 pa-tients without metastasis.Conclusion: The presence of AFPmRNA in peripheralblood may be an indicator of malignant hepatocytes,which might predict hematogenous spreading metasta-sis of tumor cells in patients with HCC.
文摘An easy and reliable method was developed for construction and quantification of competitive templates, which shared the same sequence as the amplified target DNA except for a 20 bp insertion in the middle by recombinant polymerase chain reaction (PCR). Among the advantages of competitive PCR is that any predictable or unpredictable variable that affects amplification has the same effect on both target and competitor species and that the final ratio of amplified products reflects exactly the initial targets. The utilization of a thermostable reverse transcriptase in the RT step was proposed to overcome the problem of the efficiency of target cDNA synthesis. In addition, to obtain reliable measurements, it was recommended to perform four PCR with amounts of competitive template flanking the concentration of the target mRNA.
基金This project was supported by the Washington State University Start-up Funds, George W. Bagby Research Fund
文摘Quantitative real-time reverse transcription-polymerase chain reaction （qPCR） is widely used to investigate transcriptional changes following experimental manipulations to the nervous system. Despite the widespread utilization of qPCR, the interpretation of results is marred by the lack of a suitable reference gene due to the dynamic nature of endogenous transcription. To address this inherent deficiency, we investigated the use of an exogenous spike-in mRNA, luciferase, as an internal reference gene for the 2ct normalization method. To induce dynamic transcription, we systemically administered capsaicin, a neurotoxJn selective for C-type sensory neurons expressing the TRPV-1 receptor, to adult male Sprague-Dawley rats. We later isolated nodose ganglia for qPCR analysis with the reference being either exogenous luciferase mRNA or the commonly used endogenous reference 13-111 tubulin. The exogenous luciferase mRNA reference clearly demonstrated the dynamic expression of the endogenous reference. Furthermore, variability of the endogenous reference would lead to misinterpretation of other genes of interest. In conclusion, traditional reference genes are often unstable under physiologically normal situations, and certainly unstable following the damage to the nervous system. The use of exogenous spike-in reference provides a consistent and easily implemented alternative for the analysis of qPCR data.
文摘In order to detect circulating cells of hepatocellular carcinoma(HCC) in the peripheral blood with reverse transcripition polymerase chain reaction (RT-PCR ), alpha-fetoprotein (AFP ) mRNA was tested in the blood samples of 113 cases of HCC and 69 controls (including 30 cases of liver cirrhosis, 9 cases of metastatic liver cancer and 30 normal subjects). 20/43 (46. 5% ) cases of HCC and 2/30 (6. 7% ) cases of liver cirrhosis are positive and the cases of nletastatic liver cancer and normal controls were negative for human AFP(hAFP) rnRNA. The presence of hAFP mRNA in the peripheral blood seems to be correlated with intrahepatic and distant nletastasls of HCC and portal vein thrombosis. It is concluded that the presence of hAFP mRNA in the peripheral hloocl is an indicator of circulating HCC cells and can be used to diagnose the rnetastasisof HCC through henlatogenous route and RT-PCR amplification of hAFP mRNA is a sensitive and specificprocedure for detecting circulating cells of HCC.
基金Supported by postdoctoral research fellowship from FIGO/ESRF (International Federation of Gynecology and Obsterics/Ernst Schering Research Foundation).
文摘Objective： To explore an ideal approach for detecting the physical status of HPV-16 in clinic use and to investigate the integrated HPV-16 in CINs and cervical cancer. Methods： Multiplex real-time PCR method was established to quantify the copy numbers of E2 and E6 genes （E2/E6） for analysis of the physical status of HPV-16 DNA and this assay was compared to Southern blot analysis. HPV-16-containing paraffin-embedded tissues including 49 CINs and 51 cervical squamous cancers were detected using the method. Results： （1） The cutoff ratio of E2/E6 to distinguish pure episomal from mixed HPV-16, was 0.81 in the multiplex real-time PCR; （2） The agreement rate between multiplex real-time PCR and Southern blot was 81.5% （the Kappa statistic was 0.844, P〈0.001）; （3） HPV-16 DNA existed in an episomal form in 57.1% and mixed form in 42.9% of CIN I lesions; The concomitant form of HPV-16 （〉70%） constituted the majodty in CIN Ⅱ and CIN Ⅲ; HPV-16 DNA mostly integrated into the host chromosome （s） in squamous cervical cancers （68.6%）; （4） The incidence of HPV-16 integration was increased with the degree of cervical lesions; （5） The frequency of pure integrated HPV-16 in stage Ⅱ＋Ⅲ （88%） was significantly higher than that in stage Ⅰ （33.3%）. Conclusion： （1） Mutiplex real-time PCR provides a rapid, sensitive and reliable method for clinic detection of the physical state of HPV-16 DNA; （2） The integration of the HPV-16 DNA is a very eady and important event in the progression from preinvasive to invasive cervical cancer; （3） The pure integrated status of HPV-16 in cervical cancer may be associated with poor prognosis of cervical cancer, but further study will be needed to prove its prognostic significance.
文摘Objective To establish a multiplex polymerase chain reaction （M-PCR） assay for simultaneous detection of pathogens causing genital ulcer disease （GUD）. Mothods Based on the gene-specific region of the following pathogens： Chlamydia trachomatis omp l/ompb, herpes simplex virus （HSV） DNA polymerase, Treponema pollidum tpp47, Haemophilus ducreyi 16s rRNA, four sets of primers were designed and an M-PCR assay was developed to detect four pathogens in one test. The assay was evaluated with diagnostic result of golden standard for each pathogen.Results Of the 51 clinical samples, M-PCR showed slightly higher positive rate （47.1%） of HSV than cell culture （23.6%）. Meanwhile, the positive rate of T. pallidum detected by M-PCR and dark-field microscopy was 19.6% （10/51） and 15.7% （8/51）, respectively. Only one sample was positive for H. ducreyi and no sample was positive for C. trachomatis detected by both M-PCR assay and culture. Conclusion This primary study indicated that M-PCR assay can simultaneously and rapidly detect the four etiologic pathogens causing GUD.
文摘BACKGROUND Recently,stool multiplex polymerase chain reaction(PCR)tests have been developed for identifying diarrhea-causing bacterial pathogens.Furthermore,fecal calprotectin is a well-known effective marker for intestinal mucosal inflammation.AIM To evaluate the efficacy of stool multiplex PCR and fecal calprotectin in acute infectious diarrhea.METHODS Overall,400 patients with acute infectious diarrhea were enrolled from Kangdong Sacred Heart Hospital(January 2016 to December 2018).Multiplex PCR detected 7 enteropathogenic bacteria including Salmonella,Campylobacter,Shigella,Escherichia coli O157:H7,Aeromonas,Vibrio,and Clostridium difficile.We reviewed clinical and laboratory findings using stool multiplex PCR.RESULTS Stool multiplex PCR test detected considerably more bacterial pathogens than stool culture(49.2%vs 5.2%),with Campylobacter as the most common pathogen(54%).Patients with positive stool PCR showed elevated fecal calprotectin expression compared to patients with negative stool PCR(1124.5±816.9 mg/kg vs 609±713.2 mg/kg,P=0.001).C-reactive protein(OR=1.01,95%CI:1.001-1.027,P=0.034)and sigmoidoscopy-detected colitis(OR=4.76,95%CI:1.101-20.551,P=0.037)were independent factors in stool PCR-based detection of bacterial pathogens.Sensitivity and specificity of calprotectin were evaluated to be 70.5%and 60.9%,respectively(adjusted cut-off value=388 mg/kg).CONCLUSION Stool multiplex PCR test has increased sensitivity in detecting pathogens than conventional culture,and it is correlated with calprotectin expression.Stool multiplex PCR and calprotectin may be effective in predicting clinical severity of infectious diarrhea.
文摘OBJECTIVE: To assess the feasibility of detecting SYT-SSX fusion transcripts in paraffin-embedded tissues of synovial sarcoma by reverse transcription-polymerase chain reaction (RT-PCR). METHODS: RT-PCR was used to amplify the SYT-SSX fusion transcripts using archival formalin-fixed paraffin-embedded tumor specimens from a series of 37 synovial sarcoma cases. To investigate the specificity of the SYT-SSX fusion transcripts, a variety of non-synovial sarcoma tumors were included in the study as negative controls. The detected messages derived from fusion genes were confirmed by subsequent sequence analysis. RESULTS: SYT-SSX fusion transcripts were detected in 33 of 37 (89.2%) synovial sarcomas. None of the 34 cases of non-synovial sarcoma tumors showed amplified products of SYT-SSX fusion transcripts, although PBGD mRNA was detected in all specimens. Among 33 SYT-SSX-positive synovial sarcomas, 22 tumors had an SYT-SSX 1 fusion transcript, whereas 6 tumors had an SYT-SSX2 fusion transcript. Fusion types can not be distinguished in the remaining 5 cases. There was a significant relationship between SYT-SSX fusion type and histologic subtype. All 10 biphasic synovial sarcomas had the SYT-SSX1 fusion, whereas all tumors with SYT-SSX2 were of monophasic morphology (P
基金supported by the National Nature Science Foundation of China,Grant Number:81400639the Science Foundation for Youth Scientists of the Second People’s Hospital of Guangdong Province of China,Grant Number:YQ2015-002
文摘Objective: To access the performance of the Tellgenplex human papillomavirus(HPV) DNA test compared to the polymerase chain reaction-reverse dot blot(PCR-RDB) assay for the HPV genotyping.Methods: Sixty cervical swab samples were genotyped by the Tellgenplex HPV DNA test and the PCR-RDB assay.The Tellgenplex HPV DNA test and the PCR-RDB assay can detect 26 and 23 HPV genotypes, respectively.Each sample showed discrepancy was genotyped using sequencing.Results: The percent agreement between the two tests ranged from 83.3% to 100.0% according to different genotype.This showed perfect agreement(>0.81) for high-risk HPV genotypes(35, 39, 45, 53, 56, 59, 66, 68, and 82), substantial agreement(>0.65) for high-risk HPV genotypes(16, 18, 33, 52, and 58) and low-risk HPV genotype 43 between the two assays by the kappa analysis.The positive rates of the two assays for frequent HPV genotypes(16, 35, 39, 45, 52, 53, 58, 59, 66, and 82) were not statistically different, but the PCR-RDB assay showed higher positive rates than the Tellgenplex HPV DNA test for HPV genotypes 81(P<0.05).As for more than 10 positive results by the Tellgenplex HPV DNA test and/or the PCR-RDB assay, the PCR-RDB assay showed higher relative sensitivity and specificity than the Tellgenplex HPV DNA test for the three HPV genotypes(16, 52, and 81).All HPV genotypes that can be detected by only the Tellgenplex HPV DNA test(HPV genotypes 44 and 55) were confirmed by sequencing.Conclusions: In conclusion, our results demonstrated that the PCR-RDB assay which can detect more multiple HPV genotypes in each specimen shows higher relative sensitivity and specificity than the Tellgenplex HPV DNA test, which makes it a better option for routine clinical use.
文摘A set of allele-specific oligonucleotide (ASO) probes used for detecting all 18 β-tha-lassemia mutations found in Chinese was immobilized on two strips of Biodyne C membrane;one containing 7 pairs of oligonucleotide probes specific for the most commonly found mutant al-leles,and the other containing the remaining 11 pairs of ASO_s specific for the less commonlyfound.The membranes were hybridized with β-globin sequences amplified by polymerase chainreaction (PCR) with biotinylated primers,and then treated with Streptavidin-POD conjugateand substrates for color development.The method has been applied successfully to the detectionof all 18 Chinese β-thalassemia mutations and prenatal diagnosis of two high-risk pregnancies ofβ-thalassemia.Patients with homozygous,heterozygous and compound heterozygous alleles ofthese mutations and normal individuals could be easily distinguished by the present method.Us-ing the immobilized-probe format (reverse dot blot),it was able to screen simultaneously multi-ple β-thalassemia mutations of a DNA sample by performing hybridization only once.This assayis simple,rapid and independent of radio-isotopes and can be appplied for all 18 β-thalassemiamutations so far found in Chinese population.It is considered that this method may be usefulfor gene frequency investigation of large numbers of β-thalassemia DNA samples and used as aroutine method in the clinic laboratory.
文摘SRY (sex-determining region Y chromosome) is considered as a strong candidste for the TDF (testis determining factor) and has been cloned following another candidate ZFY (zinc finger protein gene). In this study,eight cases of sex revelsal, including four 46, XX males and four 46, XY females aere examined for the presence of SRY sequence and a Y-repeated DNA locus. Our data indicated that the genownic DNA of the four classical 46,XX males had the SRY sequences. On the other hand, both SRY sequences and Ye repeated DNA sequences were present in all four 46, XY females.These resultssuavest that SAY sequences were responsible for the sexraversal of 46, XX males Whereas there may be other genetic mechanisms forthe sex reversal of 46, XY females Without the lack of SRY sequences.
文摘GBV C/HGV is a newly identified virus associated with human hepatitis In this study, the nucleotide sequences of the partial NS5 gene of GBV C/HGV derived from sera of 8 Chinese patients were determined The nucleotide homology among the 8 isolates were 92% on average On the basis of sequence analysis, two sets of oligonucleotide primers derived from highly conserved region of GBV C/HGV NS5 gene were designed to establish both sensitive and specific nested PCR for detection of GBV C/HGV RNA 253 Chinese patients were examined for the virus RNA GBV C/HGV RNA positive rates in patients infected with HBV, HCV and patients with chronic non B,non C hepatitis were 18 4%, 19 8% and 8 9% respectively This result suggested that HBV,HCV and GBV C/HGV shared the same transmission risk factors 8 patients with GBV C/HGV and HCV coinfection were retrospectively observed for the response to interferon Coinfection with GBV/HGV did not negatively influence the responsiveness of HCV, and GBV C/HGV was sensitive to interferon to a certain degree
文摘A reproducible,quantitative,non-radioactive method for the analysis of mRNA expression is described.After RNA preparation and cDNA synthesis,the cDNA was co-amplified with an internal standard in the same PCR system.The PCR products containing both targen and internal standard amplificates were electrophoresed and detected on an ABI 377 DNA Sequencer.For each sample,β-actin was also quantified by an identical procedure to compensate for relative differences between samples in the integrity of the individual RNA samples and for variations in reverse transcription.Due to the linear relationship between cDNA content and PCR product ratio of target cDNA template and competitive standard,a single PCR reaction was sufficient for quantification of a sample.The experimental results showed that the method is a mRNA quantitative RT-PCR method with high sensitivity and good reproducibility.It can be used in large-scale accurate quantitative analyses of mRNA expression of any gene.
基金This work was supported by a grant from the Department of Science and Technology of Sichuan province,China(No.30504010332).
文摘Background:Patients carrying the HongKongαα(HKαα)allele and-α3.7/αααanti-4.2 could be misdiagnosed as-α3.7/ααby the current conventional thalassemia detection methods,leading to inaccurate genetic counseling and an incorrect prenatal diagnosis.This study was aimed to accurately analyze the genotypes of HKααcarriers and-α3.7/αααanti-4.2.Methods::Samples were collected in our hospital from July 2017 to October 2019.Twenty-four common types of Chinese thalassemia were screened by gap-polymerase chain reaction(Gap-PCR)and reverse dot blot(RDB).Anti-4.2 multiplex-PCR was used to confirm carriers of theαααanti-4.2 duplication with-α3.7 deletion.Two-round nested PCR and multiplex ligation-dependent probe amplification(MLPA)were applied to accurately identify and confirm their genotypes.For data analysis,we used descriptive statistics and Fisher’s exact tests.Results::Two thousand five hundred and forty-four cases were identified as thalassemia in 5488 peripheral blood samples.The results showed thatα,β,andαβcompound thalassemia were identified in 1190(46.78%),1286(50.55%),and 68(2.67%)cases,respectively.A total of 227 samples from thalassemia patients were identified as-α3.7/ααby Gap-PCR,and the genotypes of two samples were uncertain.There was a difference between Gap-PCR and combined groups(Gap-PCR combined with nested PCR and MLPA)in detecting HKαα(P<0.05).Among the 229 patients,20 patients were identified as HKααcarriers and one was identified as-α3.7/ααα anti-4.2 by two-round nested PCR and MLPA,including 15 patients with HKαα/αα,three with HKαα/αα and β-thalassemia coinheritance,one with HKαα/-SEA,one with HKαα/-α4.2 andβ-thalassemia coinheritance,and one with-α3.7/αααanti-4.2 and β-thalassemia coinheritance.Conclusions::αααanti-4.2 and HKααgenotypes of patients carrying-α3.7 need to be detected to reduce the misdiagnosis rate of patients carrying HKααand-α3.7/αααanti-4.2 alleles.More accurate genetic counseling can be provided in the clinic using nested PCR combined with MLPA.
基金This work was supported by grants from the National Natural Science Foundation of China (No. 30200107) as well as fund from the Chenguang Plan of Wuhan City (No. 20025001027).
文摘Trisomy 21, also named Down syndrome was the most frequent autosomal aneuploidy and the most common cause of mental retardation. Fifty percent patients had congenital heart malformation. Every 20 minutes one case of trisomy 21 was born, and the incidence rate was 1 in 600 to 800 newborns in China.1 In two thirds of cases with trisomy 21, there was a spontaneous abortion, so the actual incidence was higher than that obtained postnatally.
文摘Among different endangered animal species,snakes are the most neglected creature looked at with apathy and therefore,are ruthlessly killed,illegally trafficked,and poached for their venom,lucrative skin,meat,and bones for manufacturing of medicines,accessories,and food items.Establishing the identity of the endangered snake species is important for punishing the offenders under Wildlife Protection Act(WPA)(1972)but morphological characters fail to establish identity as they are often altered.The technique of identification of snake species at molecular level holds very effective conclusion in punishing offender.Here,we have constructed and demonstrated a novel multiplexing polymerase chain reaction technique,using 16S rRNA and C-mos gene for identification of four Indian snake species,namely Ptyas mucosa,Daboia russellii,Naja naja,and Xenochrophis piscator.They are listed in Appendix-II and III of convention on international trade in endangered species of wild fauna and flora and Schedule II;Part II of Indian WPA,1972.Therefore,it may be considered a functional tool for establishing species-specific identity of four Indian snake species and promising to be useful for their conservation.
