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

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.

DETECTION OF PATHOGENS CAUSING GENITAL ULCER DISEASE BY MULTIPLEX POLYMERASE CHAIN REACTION

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.

Efficacy of stool multiplex polymerase chain reaction assay in adult patients with acute infectious diarrhea

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.

Rapid prenatal diagnosis of trisomy 21 by fluorescent quantitative multiplex polymerase chain reaction

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.

Forensic Identification of Four Indian Snake Species Using Single Multiplex Polymerase Chain Reaction

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.

Human papillomavirus 16 physical status detection in preinvasive and invasive cervical carcinoma by multiplex real-time polymerase chain reaction

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.

Dual-priming oligonucleotide-based multiplex PCR using tissue samples in rapid urease test in the detection of Helicobacter pylori infection

AIM: To investigate whether tissue samples processed by the rapid urease test (RUT) kit are suitable for dual-priming oligonucleotide-based multiplex polymerase chain reaction (DPO-PCR) to detect Helicobacter pylori (H. pylori).

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

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.

Clinical Application of Multiplex PCR Assay for the Diagnosis of the Etiology of Genital Ulcer Disease Among Patients Attending STD Clinics in Guangzhou, China

Objectives: To develop a method of simultaneous PCR detection of Haemophilus ducreyi, Treponema pallidum, andHerpes Simplex Virus Types 1 and 2 from genital ulcersamong patients attending STD clinics in Guangzhou, China;and evaluate the clinical application of multiplex PCR (M-PCR) assay for diagnosing the etiology of genital ulcerdiseases (GUD). Methods: 244 patients with a genital ulcer were evaluated.Clinical etiology of GUD was based on physical appearanceand microbiologic evaluations that included darkfieldmicroscopy examination (D-F) and serology test for syphilis(STS). Swabs of each genital ulcer were tested for HSVantigen by enzyme immunoassay (EIA) and processed in anM-PCR assay for simultaneous detection of T pallidum, HSVand H ducreyi. Results: The standard strains of T pallidum, HSV and Hducreyi were amplified by M-PCR, producing amplifiedproducts of 260bp,432bp,170bp, respectively. The sensitivityof M-PCR is 10~2 pg DNA. M-PCR assay for T.pallidum, HSVand H ducreyi showed good agreement en compared with D-F detection for T pallidum, STS, H ducreyi culture and EIAfor HSV antigen (Kappa scores are 0.774,0.704,0.793,0.756,respectively). Conclusions: The M-PCR is a convenient, accurate andreliable assay for the detection of T pallidum, HSV and Hducreyi from genital ulcers, and can be used as a method ofdiagnosing the etiology of GUD.

Multiplex RT-PCR-based detections of CEA, CK20 and EGFR in colorectal cancer patients

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.

Diagnostic performance of a multiplexed gastrointestinal PCR panel for identifying diarrheal pathogens in children undergoing hematopoietic stem cell transplant

Background Diarrhea is a common complication of hematopoietic stem cell transplantation(HSCT)and is associated with substantial morbidity,but its etiology is often unknown.Etiologies of diarrhea in this population include infectious causes,chemotherapy-or medication-induced mucosal injury and graft-versus-host disease(GVHD).Distinguishing these potential causes of diarrhea is challenging since diarrheal symptoms are often multifactorial,and the etiologies often overlap in transplant patients.The objectives of this study were to evaluate whether the FilmArray gastrointestinal(GI)panel would increase diagnostic yield and the degree to which pre-transplantation colonization predicts post-transplantation infection.Methods From November 2019 to February 2021,a total of 158 patients undergoing HSCT were prospectively included in the study.Stool specimens were obtained from all HSCT recipients prior to conditioning therapy,28±7 days after transplantation and at any new episode of diarrhea.All stool samples were tested by the FilmArray GI panel and other clinical microbiological assays.Results The primary cause of post-transplantation diarrhea was infection(57/84,67.86%),followed by medication(38/84,45.24%)and GVHD(21/84,25.00%).Ninety-five of 158 patients were colonized with at least one gastrointestinal pathogen before conditioning therapy,and the incidence of infectious diarrhea was significantly higher in colonized patients(47/95,49.47%)than in non-colonized patients(10/63,15.87%)(P<0.001).Fourteen of 19(73.68%)patients who were initially colonized with norovirus pre-transplantation developed a post-transplantation norovirus infection.Twenty-four of 62(38.71%)patients colonized with Clostridium difficile developed a diarrheal infection.In addition,FilmArray GI panel testing improved the diagnostic yield by almost twofold in our study(55/92,59.78%vs.30/92,32.61%).Conclusions Our data show that more than half of pediatric patients who were admitted for HSCT were colonized with various gastrointestinal pathogens,and more than one-third of these pathogens were associated with post-transplantation diarrhea.In addition,the FilmArray GI panel can increase the detection rate of diarrheal pathogens in pediatric HSCT patients,but the panel needs to be optimized for pathogen species,and further studies assessing its clinical impact and cost-effectiveness in this specific patient population are also needed.

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

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.

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

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.

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

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.

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

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.

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

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 assay.We investigated a pooling strategy in diagnosis of Angiostrongylus canto-nensis in Pomacea canaliculata.Two settings of speci-mens were prepared,divided into portions and detected by multiplex PCR.Specimens 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 portions.Specimens 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 portions.Repeated sampling was performed and sample size-accumulated positive rate curves were drawn.According 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 size.These test characteristics and the relevant factors to the sample size would need to be determined in much larger studies and more appropriately infield populations.The result indicates that the number of larval node is not the most important,nor the only factor to the sample size.It also implies the feasibility to detect A.cantonensis in P.canaliculata by pooling strategies.

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

Objective:To investigate the prevalence of Helicobacter pylori(H.pylori)infection in dyspepsia patients and its relation to virulence factor cagA gene.Methods: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 ureC.H.pylori was detected in 14 gastric biopsies(13%).Significantly higher numbers of female were infected.Furthermore,cag A gene was found in all H.pylori-positive specimens.In addition,the result indicated that the multiplex PCR with annealing temperature at 57℃was able to effectively amplify specific products.Conclusions:The results confirmed high prevalence of cagA gene in H.pylori among dyspeptic patients in Southern Thailand.

Developmental Validation of the EX16+22Y System

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.

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

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 minorities.In total,37 SNP-short tandem repeat haplotypes were detected.Nine of these 37 haplotypes were unique(24.32%).The gene diversity and discrimination capacity values are 0.8321 and 0.2606,respectively.Furthermore,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’samples.The 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 minorities.Similar results were shown for allele 11C and 10C.Therefore,the alleles of DYF371 with SNP C-type have the potential to differentiate the Han and non-Han Chinese populations.