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Development of a multiplex polymerase chain reaction assay for detection of hepatitis C virus,hepatitis B virus,and human immunodeficiency virus 1

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World Journal of Virology 2024年第1期13卷 95-106页

作者： Waleed Abdelgaber Nemr Radwan K Nashwa Department of Radiation Microbiology National Center for Radiation Research and TechnologyEgyptian Atomic Energy AuthorityCairo 11371Egypt Department of Health Radiation Research National Center for Radiation Research and TechnologyEgyptian Atomic Energy AuthorityCairo 11371Egypt

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 *** diagnostic methodologies that depend on the detection of viral nucleic acids are much more expensive,but they are more accurate than sero-logical *** To develop a rapid,cost-effective,and accurate diagnostic multiplex polymerase chain reaction(PCR)assay for simultaneous detection of HCV,HBV,and *** 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 ***,this diagnostic method will be appropriate for application in both conventional(combined with electrophoresis)and real-time PCR *** in silico investigations were conducted to prove the capability of the approached PCR assay to detect variants of each ***,Egyptian isolates of each virus were subjected to the wet lab examination using the given diagnostic *** The in silico investigations confirmed that the PCR primers can match many viral variants in a multiplex PCR *** wet lab experiment proved the efficiency of the assay in distinguishing each viral type through high-resolution melting *** to related published assays,the proposed assay in the current study is more sensitive and competitive with many expensive PCR *** 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.

关键词： Diagnosis Blood-borne viruses multiplex polymerase chain reaction High-resolution melting

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Molecular Analysis-Based Genetic Characterization of a Cohort of Patients with Duchenne and Becker Muscular Dystrophy in Eastern China

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Chinese Medical Journal 2018年第7期131卷 770-775页

作者： Hui-Hui Zhao Xue-Ping Sun Ming-Chao Shi Yong-Xiang Yi Hong Cheng Xing-Xia Wang Qing-Cheng Xu Hong-Ming Ma Hao-Quan Wu Qing-Wen Jin Qi Niu Oepartment of Neurology The First Affiliated Hospital of Naniing Medical University Nanjing Jiangeu 210029 China Department of Gynecology and Obstetrics The First Affiliated Hospital of Nanjing Medical University Nanjing Jiangsu 210029 China Department of Neurology The Affiliated Sir Run Run Hospital of Nanjing Medical University Nanjing Jiangsu 211166 China Department of Neurology The Second Hospital of Nanjing Nanjing Jiangsu 210003 China Department of Neurology Nanjing First Hospital Nanjing Jiangsu 210012 China Department of Biomedical Sciences Texas Tech University Health Science Center Texas 79430 USA

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.

关键词： Becker Muscular Dystrophy Duchenne Muscular Dystrophy Dystrophin multiplex Ligation-dependent Probe Amplification multiplex polymerase chain reaction Prenatal Diagnosis

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Molecular study on Y chromosome microdeletions in Egyptian males with idiopathic infertility

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Asian Journal of Andrology 2004年第1期6卷 53-57页

作者： Mostafa K. El Awady Sohair F. El Shater Ehab Ragaa Khaled Atef Ibrahim M. Shaheen Nagwa A. Megiud Department of Biomedical Technology Children with Special Needs National Research Center Dokki Cairo 12622 EgyptDepartment of Biomedical TechnologyChildren with Special Needs National Research Center Dokki Cairo 12622 EgyptDepartment of Research on Children with Special Needs National Research Center Dokki Cairo 12622 EgyptDepartment of Biomedical TechnologyChildren with Special Needs National Research Center Dokki Cairo 12622 EgyptDepartment of Dermatology Venereology and Andrology Faculty of Medicine Al-Azhar University Cairo EgyptDepartment of Research on Children with Special Needs National Research Center Dokki Cairo 12622 Egypt

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.

关键词： AZF Y microdeletions multiplex polymerase chain reaction male infertility azoospermia

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Screening for Y chromosome microdeletions in idiopathic and nonidiopathic infertile men with varicocele and cryptorchidism

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Chinese Medical Journal 2005年第17期118卷 1462-1467页

作者： SONG Ning-hong WU Hong-fei ZHANG Wei ZHUO Zuo-min QIAN Li-xing HUA Li-xing GUO Lin FENG Ning-han Department of Urology First Affiliated Hospital Nanjing Medical University Nanjing 210029 China Key Laboratory of Reproductive Medicine of Jiangsu Province Nanjing Medical University Nanjing 210029 China Model Animal Research Center Nanjing University Nanjing 210032 China

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.

关键词： Y chromosome microdeletions multiplex polymerase chain reaction male infertility

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Prevalent false positives of azoospermia factor a （AZFa） microdeletions caused by single-nucleotide polymorphism rs72609647 in the sY84 screening of male infertility

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Asian Journal of Andrology 2011年第6期13卷 877-880页

作者： Qing Wu Guo-Wu Chen Tao-Fei Yan Hui Wang Yu-Ling Liu Zheng Li Shi-Wei Duan Fei Sun Yun Feng Hui-Juan Shi Reproductive Medical Center Ruijin Hospital Shanghai Jiao Tong University School of Medicine Shanghai 200025 China Shanghai Jiai Genetics and IVF Institute Center of China-USA Shanghai 200011 China Department of Obstetrics and Gynecology Second Military Medical University Shanghai 200433 China China National Population and Family Planning Key Laboratory of Contraceptive Drugs and Devices Shanghai Institute of Planned Parenthood Research Shanghai 200032 China Shanghai Human Sperm Bank Shanghai Institute of Andrology Renji Hospital Shanghai Jiao Tong University School of Medicine Shanghai 200001 China School of Medicine Ningbo University Ningbo 315033 China Laboratory for Reproductive Biology School of Life Science University of Science and Technology of China Hefei 230026 China

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.

关键词： male infertility multiplex polymerase chain reaction rs72609647 single-nucleotide polymorphism sY84 Y-chromosomemicrodeletion

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A pooling strategy of a PCR-based assay to detectAngiostrongylus cantonensis in snail intermediate host,Pomacea canaliculata

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Frontiers of Medicine 2010年第2期4卷 204-207页

作者： Fu-Rong WEI Shan LV He-Xiang LIU Ling HU Yi ZHANG Department of Vector Control National Institute of Parasitic DiseaseChinese Center for Disease Control and PreventionKey Laboratory of Parasite and Vector Biology of the Ministry of HealthShanghai 200025China

Poolingﬁeld specimens could reduce the number of assay and thus increase the efﬁciency in detecting and screening pathogen infections by polymerase chain reaction(PCR)-based *** investigated a pooling strategy in diagnosis of Angiostrongylus canto-nensis in Pomacea *** settings of speci-mens were prepared,divided into portions and detected by multiplex *** A was 0.4490 g positive lung tissue of 28 larval nodes from four snails mixed with 1.310 g negative lung tissue from six snails and divided into 32 *** B was 0.5448 g positive lung tissue with 26 larval nodes from two snails mixed with 1.092 g negative lung tissue from seven snails and divided into 48 *** sampling was performed and sample size-accumulated positive rate curves were *** to the sample size-accumulated positive rate curves,the appropriate sample size of the two specimens were 18 and 15,respectively,which is 0.36–0.58 to the total sample *** test characteristics and the relevant factors to the sample size would need to be determined in much larger studies and more appropriately inﬁeld *** result indicates that the number of larval node is not the most important,nor the only factor to the sample *** also implies the feasibility to detect *** in *** by pooling strategies.

关键词： Angiostrongylus cantonensis Pomacea cana-liculata multiplex polymerase chain reaction diagnosis pooling

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Establishment of a multiplex PCR System to Diagnose Tuberculosis and Other Bacterial Infections

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Journal of Huazhong University of Science and Technology(Medical Sciences) 2000年第4期20卷 324-326页

作者：方峰向稚丹陈如 Department of Pediatrics Tongji Hospital Tongji Medical University Wuhan Department of Laboratory Medicine Tongji Hospital Tongji Medical University Wuhan

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.

关键词： 16S rRNA gene multiplex polymerase chain reaction bacterium tubercle bacillus

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Developmental Validation of the EX16+22Y System

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Journal of Forensic Science and Medicine 2020年第3期6卷 79-88页

作者： Xin Wang Weizhong Chen Jian Zhang Yang Cui Jingjing Chen Yunjie Shi Gao Wang Fayuan Li Yong Liu Ruhua Zhou Department of DNA Laboratory Institute of Criminal Science and TechnologySuzhou Municipal Public Security BureauXiangchengSuzhouChina Department of Research and Development AGCU ScienTech IncorporationHuishanWuxiChina

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 *** 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 *** 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 *** results show that the EX16+22Y system is a robust and reliable amplification kit which can be used for human identification testing.

关键词： DNA typing forensic science multiplex polymerase chain reaction short tandem repeat validation

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A Potential Marker DYF371 for Differentiating Han Population from Non-Han Population in Chinese

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Journal of Forensic Science and Medicine 2020年第1期6卷 1-4,I0001页

作者： Hailun Nan Dejian Lu Faculty of Forensic Medicine Zhongshan Medical SchoolSun Yat‑Sen UniversityGuangzhouChina

DYF371 locus and single‑nucleotide polymorphisms(SNPs)defined as DYF371C or DYF371T were simultaneously examined for 142 Chinese unrelated male individuals,including 90 Han population and 52 *** total,37 SNP-short tandem repeat haplotypes were *** of these 37 haplotypes were unique(24.32%).The gene diversity and discrimination capacity values are 0.8321 and 0.2606,***,only five males(5.55%)in the Han samples were determined that they had one copy of allele with SNP T-type and three copies of alleles with SNP C-types,whereas 14 individuals(26.92%)were observed in minorities’*** genotype proportion comprising three distinguishing copies of the allele with SNP C-types in Han sample was greatly lower than that in the sample of *** results were shown for allele 11C and ***,the alleles of DYF371 with SNP C-type have the potential to differentiate the Han and non-Han Chinese populations.

关键词： DYF371 multi-copy multiplex polymerase chain reaction single-nucleotide polymorphism polymorphism-short tandem repeat

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Prevalence of Helicobacter pylori cagA genotype among dyspeptic patients in Southern Thailand

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Journal of Coastal Life Medicine 2014年第11期 895-898页

作者： Sueptrakool Wisessombat Chatruthai Meethai School of Allied Health Sciences and Public Health Walailak UniversityTha SalaNakhon Si Thammarat ProvinceThailand Faculty of Medical Technology Prince of Songkla UniversityHat YaiSongkhla ProvinceThailand

Objective:To investigate the prevalence of Helicobacter pylori(***)infection in dyspepsia patients and its relation to virulence factor cagA ***:In total,110 gastric biopsies from dyspeptic patients were comparatively studied using rapid urease test and multiplex polymerase chain reaction(PCR).Results:multiplex PCR detected three genes of 16S rRNA,cagA,and *** was detected in 14 gastric biopsies(13%).Significantly higher numbers of female were ***,cag A gene was found in all ***-positive *** addition,the result indicated that the multiplex PCR with annealing temperature at 57℃was able to effectively amplify specific ***:The results confirmed high prevalence of cagA gene in *** among dyspeptic patients in Southern Thailand.

关键词： Helicobacter pylori multiplex polymerase chain reaction Dyspepsia cagA gene

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