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
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
In order to rapidly diagnose and differentiate tuberculosis from other bacterial infections, a 16S rRNA gene (16s rDNA)-directed multiplex PCR system was developed. In this system, a pair of universal primers and a ...In order to rapidly diagnose and differentiate tuberculosis from other bacterial infections, a 16S rRNA gene (16s rDNA)-directed multiplex PCR system was developed. In this system, a pair of universal primers and a tubercle bacillus (Tb)-specific primer were designed based on highly con- served regions and Tb species-specific variable region of bacterial 16s rDNA. A 360bp fragment was detected in all bacteria tested, and a 210bp fragment was found only in Tb. 19 species of known bac- teria including Tb were used for evaluating specificity, universality and sensitivity of the PCR. Candi- da albicans and human diploid cell served as controls. It was found that both 210bp and 360bp frag- ments were amplified only in Tb, and only 360 bp fragment was detected in other 18 species of gener- al bacteria. Candida albicans and human cells were negative for both 360bp and 2l0bp fragments. The lowest detectable level of the PCR was 10 fg of DNA for Escherichia coli and 100 fg of DNA for Tb. The results indicated that this multiplex PCR system for the simultaneous detection of Tb and other common bacteria had higher specificity and sensitivity, as well as good universality and might be useful to rapidly diagnose bacterial infections and effectively distinguish tuberculosis from other bacterial involvement.展开更多
AIM: To develop a multiplex reverse transcription polymerase chain reaction (RT-PCR) method detecting cir-culating tumor cells in the peripheral blood of colorectal cancer (CRC) patients. METHODS: Peripheral blood sam...AIM: To develop a multiplex reverse transcription polymerase chain reaction (RT-PCR) method detecting cir-culating tumor cells in the peripheral blood of colorectal cancer (CRC) patients. METHODS: Peripheral blood samples were collected from 88 CRC patients and 40 healthy individuals from the blood donors' clinic and subsequently analyzed by multiplex RT-RCR for the expression of carcinoembryonic antigen (CEA), cytokeratin 20 (CK20) and epidermal growth factor receptor (EGFR) mRNA. The analysis involved determining the detection rates of CEA, CK20 and EGFR transcripts vs disease stage and overall survival. Median follow-up period was 19 mo (range 8-28 mo). RESULTS: Rates of CEA, CK20 and EGFR detection in CRC patients were 95.5%, 78.4% and 19.3%, respectively. CEA transcripts were detected in 3 healthy volunteer samples (7.5%), whereas all control samples were tested negative for CK20 and EGFR transcripts. The increasing number of positive detections for CEA, CK20 and EGFR transcripts in each blood sample was positively correlated with Astler-Coller disease stage (P< 0.001) and preoperative serum levels of CEA (P=0.029) in CRC patients. Data analysis using Kaplan-Meier estimator documented signif icant differences in the overall survival of the different CRC patient groups as formed according to the increasing number of positivity for CEA, CK20 and EGFR transcripts. CONCLUSION: These data suggest that multiplex RTPCR assay can provide useful information concerning disease stage and overall survival of CRC 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 determine the frequency of genetic deletions within the azoospermia factors in Egyptian infertile males. Methods: The Yq microdeletions in 33 infertile males with undetectable chromosomal anomalies were examin...Aim: To determine the frequency of genetic deletions within the azoospermia factors in Egyptian infertile males. Methods: The Yq microdeletions in 33 infertile males with undetectable chromosomal anomalies were examined by mutiplex polymerase chain reaction (PCR). Deletions were confirmed using single PCR amplifications. Results: Four out of the total 33 (12 %) men had Yq11 microdeletions, thus supporting the average reported figures in other populations. Three of those 4 cases had single short tandem sequence deletions with discrete histological findings of their testes. Single sY272 deletion within AZFc was associated with Sertoli cell only syndrome, whereas a patient with isolated sY84 deletion within AZFa had immature testicular structure. The remaining case had a large deletion in AZFa-c and short stature. Conclusion: The present study supports the hypothesis that the Yqn encompasses genetic determinants of stature besides genes controlling spermatogenesis.展开更多
Nowadays the role of genetic findings in determining the diagnosis,therapy and prognosis of acute myeloid leukemia(AML) has become more valuable.To improve and validate the detection of clonal chromosomal aberrations ...Nowadays the role of genetic findings in determining the diagnosis,therapy and prognosis of acute myeloid leukemia(AML) has become more valuable.To improve and validate the detection of clonal chromosomal aberrations in leukemia,we designed a combined application of karyotyping with multiplex reverse transcription-polymerase chain reaction(RT-PCR) and fluorescence in situ hybridization(FISH),and addressed the expression and distribution of fusion genes among the subtypes of Chinese adult patients with de novo AML.Multiplex RT-PCR assays were performed on 477 samples from newly diagnosed AML patients,and cytogenetic data were obtained from 373 of them by R or G banding techniques and those in some cases were confirmed by FISH.The PCR products in some suspected cases were tested by two-directional sequencing.The results showed that except unqualified samples,fusion genes were detected by multiplex RT-PCR in 211 of 474 patients(44.51%),including AML1-ETO,CBFβ-MYH11,PML-RARα,PLZF-RARα,NPM-RARα,MLL rearrangements,BCR-ABL,DEK-CAN,SET-CAN,TEL-PDGFR,TLS-ERG,AML1-MDS1(EVI-1).In 373 patients,who took both multiplex RT-PCR and karyotype analysis,the detection rate of chromosomal aberrations by using multiplex RT-PCR and karyotyping was 160/373(42.89%) and 179/373(47.98%) respectively,and the combination could optimize the detection rate of clonal genetic abnormalities to 216/373(57.90%).The PCR results from 11 cases 'normal' in karyotyping but abnormal in RT-PCR for MLL rearrangements were confirmed by two-directional sequencing.It is concluded that karyotype studies remain the cornerstone for genetic testing;conventional cytogenetics and molecular-based methods are complementary tests for the detection of clonal genetic aberrations in AML,especially for the cryptic or submicroscopic aberrations.Once a genetic marker has been identified by combined analysis,it could be used to monitor residual disease during/after chemotherapy,by quantitative RT-PCR and/or FISH.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
The EX 16+22Y system is a polymerase chain reaction(PCR)-based amplification kit that enables typing of 15 autosomal short tandem repeat(STR)loci(i.e„D3S1358,D13S317,D7S820,D16S539,TPOX,TH01,D2S133&CSF1PO,D19S433,...The EX 16+22Y system is a polymerase chain reaction(PCR)-based amplification kit that enables typing of 15 autosomal short tandem repeat(STR)loci(i.e„D3S1358,D13S317,D7S820,D16S539,TPOX,TH01,D2S133&CSF1PO,D19S433,vWA,D18S51,D21S11,D8S1179,D5S81&and FGA)and 22 widely used Y chromosome STR(Y-STR)loci(DYS391,DYS527a/b,DYS635,DYS458,DYS456,DYS385a/b,DYS43&DYS44&DYS437,DYS19,DYS576,DYS533,DYS393,DYS389I/n,DYS439,DYS392,Y_GATA_H4,DYS390,and DYS481)which contains 20 core Y-STR recommended by the Ministry of Public Security and amelogenin.This multiplex system was designed for the simultaneous analysis of amelogenin-Y allele mutation,single-source searches,kinship(including familial searching),mixture profiles,international data sharing,and other forensic applications.In this study,the multiplex system was validated for sensitivity of detection,species specificity,DNA mixtures,stability,sizing precision,stutter,reproducibility,and PCR-based conditions according to the Scientific Working Group on DNA Analysis Methods developmental validation guidelines and Chinese criteria for the human fluorescent STR multiplex PCR reagent.The results show that the EX16+22Y system is a robust and reliable amplification kit which can be used for human identification testing.展开更多
Cytogenetic and molecular studies of azoospermic and oligozoospermic males have suggested the presence of azoospermia factors (AZF) in the Y chromosome. Deletion in AZF regions has been reported to disrupt spermatog...Cytogenetic and molecular studies of azoospermic and oligozoospermic males have suggested the presence of azoospermia factors (AZF) in the Y chromosome. Deletion in AZF regions has been reported to disrupt spermatogenesis and cause infertility. Several candidate genes responsible for spermatogenesis have been identified in this region and some of them are thought to be functional in human spermatogenesis. And we reported clinical and molecular studies of Y chromosome microdeletions in Chinese. This study aimed at assessing the frequency of microdeletions in Chinese men with idiopathic and nonidiopathic infertility problems and dicussing the clinical significance of the AZF region.展开更多
Background: Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are common X-linked recessive neuromuscular disorders caused by mutations in dystrophin gene. Multiplex polymerase chain reaction ...Background: Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are common X-linked recessive neuromuscular disorders caused by mutations in dystrophin gene. Multiplex polymerase chain reaction (multiplex PCR) and multiplex ligation-dependent probe amplification (MLPA) are the most common methods for detecting dystrophin gene mutations, This study aimed to contrast the two methods and discern the genetic characterization of patients with DMD/BMD in Eastern China. Methods: We collected 121 probands, 64 mothers ofprobands, and 15 fetuses in our study. The dystrophin gene was detected by multiplex PCR primarily in 28 probands, and MLPA was used in multiplex PCR-negative cases subsequently. The dystrophin gene of the remaining 93 probands and 62 female potential carriers was tested by MLPA directly. In fetuses, multiplex PCR and MLPA were performed on 4 fetuses and 10 fetuses, respectively. In addition, sequencing was also performed in 4 probands with negative MLPA. Results: We found that 61.98% of the subjects had genetic mutations including deletions (50.41%) and duplications (11.57%). There were 43.75% of mothers as carriers of the mutation. In 15 fetuses, 2 out of 7 male fetuses were found to be unhealthy and 2 out of 8 female fetuses were found to be carriers. Exons 3-26 and 45-52 have the maximum frequency in mutation regions. In the frequency ofexons individually, exon 47 and exon 50 were the most common in deleted regions and exons 5, 6, and 7 were found most frequently in duplicated regions. Conclusions: MLPA has better productivity and sensitivity than multiplex PCR. Prenatal diagnosis should be applied in DM D high-risk fetuses to reduce the disease incidence. Furthermore, it is the responsibility of physicians to inform female carriers the importance of prenatal diagnosis.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.
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In order to rapidly diagnose and differentiate tuberculosis from other bacterial infections, a 16S rRNA gene (16s rDNA)-directed multiplex PCR system was developed. In this system, a pair of universal primers and a tubercle bacillus (Tb)-specific primer were designed based on highly con- served regions and Tb species-specific variable region of bacterial 16s rDNA. A 360bp fragment was detected in all bacteria tested, and a 210bp fragment was found only in Tb. 19 species of known bac- teria including Tb were used for evaluating specificity, universality and sensitivity of the PCR. Candi- da albicans and human diploid cell served as controls. It was found that both 210bp and 360bp frag- ments were amplified only in Tb, and only 360 bp fragment was detected in other 18 species of gener- al bacteria. Candida albicans and human cells were negative for both 360bp and 2l0bp fragments. The lowest detectable level of the PCR was 10 fg of DNA for Escherichia coli and 100 fg of DNA for Tb. The results indicated that this multiplex PCR system for the simultaneous detection of Tb and other common bacteria had higher specificity and sensitivity, as well as good universality and might be useful to rapidly diagnose bacterial infections and effectively distinguish tuberculosis from other bacterial involvement.
基金Supported by The Ministry of Development of the Greek Government (GGET-AKMON)
文摘AIM: To develop a multiplex reverse transcription polymerase chain reaction (RT-PCR) method detecting cir-culating tumor cells in the peripheral blood of colorectal cancer (CRC) patients. METHODS: Peripheral blood samples were collected from 88 CRC patients and 40 healthy individuals from the blood donors' clinic and subsequently analyzed by multiplex RT-RCR for the expression of carcinoembryonic antigen (CEA), cytokeratin 20 (CK20) and epidermal growth factor receptor (EGFR) mRNA. The analysis involved determining the detection rates of CEA, CK20 and EGFR transcripts vs disease stage and overall survival. Median follow-up period was 19 mo (range 8-28 mo). RESULTS: Rates of CEA, CK20 and EGFR detection in CRC patients were 95.5%, 78.4% and 19.3%, respectively. CEA transcripts were detected in 3 healthy volunteer samples (7.5%), whereas all control samples were tested negative for CK20 and EGFR transcripts. The increasing number of positive detections for CEA, CK20 and EGFR transcripts in each blood sample was positively correlated with Astler-Coller disease stage (P< 0.001) and preoperative serum levels of CEA (P=0.029) in CRC patients. Data analysis using Kaplan-Meier estimator documented signif icant differences in the overall survival of the different CRC patient groups as formed according to the increasing number of positivity for CEA, CK20 and EGFR transcripts. CONCLUSION: These data suggest that multiplex RTPCR assay can provide useful information concerning disease stage and overall survival of CRC 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.
文摘Aim: To determine the frequency of genetic deletions within the azoospermia factors in Egyptian infertile males. Methods: The Yq microdeletions in 33 infertile males with undetectable chromosomal anomalies were examined by mutiplex polymerase chain reaction (PCR). Deletions were confirmed using single PCR amplifications. Results: Four out of the total 33 (12 %) men had Yq11 microdeletions, thus supporting the average reported figures in other populations. Three of those 4 cases had single short tandem sequence deletions with discrete histological findings of their testes. Single sY272 deletion within AZFc was associated with Sertoli cell only syndrome, whereas a patient with isolated sY84 deletion within AZFa had immature testicular structure. The remaining case had a large deletion in AZFa-c and short stature. Conclusion: The present study supports the hypothesis that the Yqn encompasses genetic determinants of stature besides genes controlling spermatogenesis.
文摘Nowadays the role of genetic findings in determining the diagnosis,therapy and prognosis of acute myeloid leukemia(AML) has become more valuable.To improve and validate the detection of clonal chromosomal aberrations in leukemia,we designed a combined application of karyotyping with multiplex reverse transcription-polymerase chain reaction(RT-PCR) and fluorescence in situ hybridization(FISH),and addressed the expression and distribution of fusion genes among the subtypes of Chinese adult patients with de novo AML.Multiplex RT-PCR assays were performed on 477 samples from newly diagnosed AML patients,and cytogenetic data were obtained from 373 of them by R or G banding techniques and those in some cases were confirmed by FISH.The PCR products in some suspected cases were tested by two-directional sequencing.The results showed that except unqualified samples,fusion genes were detected by multiplex RT-PCR in 211 of 474 patients(44.51%),including AML1-ETO,CBFβ-MYH11,PML-RARα,PLZF-RARα,NPM-RARα,MLL rearrangements,BCR-ABL,DEK-CAN,SET-CAN,TEL-PDGFR,TLS-ERG,AML1-MDS1(EVI-1).In 373 patients,who took both multiplex RT-PCR and karyotype analysis,the detection rate of chromosomal aberrations by using multiplex RT-PCR and karyotyping was 160/373(42.89%) and 179/373(47.98%) respectively,and the combination could optimize the detection rate of clonal genetic abnormalities to 216/373(57.90%).The PCR results from 11 cases 'normal' in karyotyping but abnormal in RT-PCR for MLL rearrangements were confirmed by two-directional sequencing.It is concluded that karyotype studies remain the cornerstone for genetic testing;conventional cytogenetics and molecular-based methods are complementary tests for the detection of clonal genetic aberrations in AML,especially for the cryptic or submicroscopic aberrations.Once a genetic marker has been identified by combined analysis,it could be used to monitor residual disease during/after chemotherapy,by quantitative RT-PCR and/or FISH.
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.
文摘The EX 16+22Y system is a polymerase chain reaction(PCR)-based amplification kit that enables typing of 15 autosomal short tandem repeat(STR)loci(i.e„D3S1358,D13S317,D7S820,D16S539,TPOX,TH01,D2S133&CSF1PO,D19S433,vWA,D18S51,D21S11,D8S1179,D5S81&and FGA)and 22 widely used Y chromosome STR(Y-STR)loci(DYS391,DYS527a/b,DYS635,DYS458,DYS456,DYS385a/b,DYS43&DYS44&DYS437,DYS19,DYS576,DYS533,DYS393,DYS389I/n,DYS439,DYS392,Y_GATA_H4,DYS390,and DYS481)which contains 20 core Y-STR recommended by the Ministry of Public Security and amelogenin.This multiplex system was designed for the simultaneous analysis of amelogenin-Y allele mutation,single-source searches,kinship(including familial searching),mixture profiles,international data sharing,and other forensic applications.In this study,the multiplex system was validated for sensitivity of detection,species specificity,DNA mixtures,stability,sizing precision,stutter,reproducibility,and PCR-based conditions according to the Scientific Working Group on DNA Analysis Methods developmental validation guidelines and Chinese criteria for the human fluorescent STR multiplex PCR reagent.The results show that the EX16+22Y system is a robust and reliable amplification kit which can be used for human identification testing.
基金The work was supported by the "135" Foundation of JiangsuProvince (No.0151).
文摘Cytogenetic and molecular studies of azoospermic and oligozoospermic males have suggested the presence of azoospermia factors (AZF) in the Y chromosome. Deletion in AZF regions has been reported to disrupt spermatogenesis and cause infertility. Several candidate genes responsible for spermatogenesis have been identified in this region and some of them are thought to be functional in human spermatogenesis. And we reported clinical and molecular studies of Y chromosome microdeletions in Chinese. This study aimed at assessing the frequency of microdeletions in Chinese men with idiopathic and nonidiopathic infertility problems and dicussing the clinical significance of the AZF region.
基金This study was supported by grants from the National Natural Science Foundation of China (No. 81671117), the Jiangsu Province Natural Science Foundation (No. BK20141439), and A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (No. JXC 10231802).
文摘Background: Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are common X-linked recessive neuromuscular disorders caused by mutations in dystrophin gene. Multiplex polymerase chain reaction (multiplex PCR) and multiplex ligation-dependent probe amplification (MLPA) are the most common methods for detecting dystrophin gene mutations, This study aimed to contrast the two methods and discern the genetic characterization of patients with DMD/BMD in Eastern China. Methods: We collected 121 probands, 64 mothers ofprobands, and 15 fetuses in our study. The dystrophin gene was detected by multiplex PCR primarily in 28 probands, and MLPA was used in multiplex PCR-negative cases subsequently. The dystrophin gene of the remaining 93 probands and 62 female potential carriers was tested by MLPA directly. In fetuses, multiplex PCR and MLPA were performed on 4 fetuses and 10 fetuses, respectively. In addition, sequencing was also performed in 4 probands with negative MLPA. Results: We found that 61.98% of the subjects had genetic mutations including deletions (50.41%) and duplications (11.57%). There were 43.75% of mothers as carriers of the mutation. In 15 fetuses, 2 out of 7 male fetuses were found to be unhealthy and 2 out of 8 female fetuses were found to be carriers. Exons 3-26 and 45-52 have the maximum frequency in mutation regions. In the frequency ofexons individually, exon 47 and exon 50 were the most common in deleted regions and exons 5, 6, and 7 were found most frequently in duplicated regions. Conclusions: MLPA has better productivity and sensitivity than multiplex PCR. Prenatal diagnosis should be applied in DM D high-risk fetuses to reduce the disease incidence. Furthermore, it is the responsibility of physicians to inform female carriers the importance of prenatal diagnosis.