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.

Locked nucleic acid real-time polymerase chain reaction method identifying two polymorphisms of hepatitis B virus genotype C2 infections,rt269L and rt269I

BACKGROUND The presence of two distinct hepatitis B virus(HBV)Pol RT polymorphisms,rt269L and rt269I,could contribute to the unique clinical or virological phenotype of HBV genotype C2.Therefore,a simple and sensitive method capable of identifying both types in chronic hepatitis B(CHB)patients infected with genotype C2 should be developed.AIM To develop a novel simple and sensitive locked nucleic acid(LNA)-real timepolymerase chain reaction(RT-PCR)method capable of identifying two rt269 types in CHB genotype C2 patients.METHODS We designed proper primer and probe sets for LNA-RT-PCR for the separation of rt269 types.Using synthesized DNAs of the wild type and variant forms,melting temperature analysis,detection sensitivity,and endpoint genotyping for LNA-RT-PCR were performed.The developed LNA-RT-PCR method was applied to a total of 94 CHB patients of genotype C2 for the identification of two rt269 polymorphisms,and these results were compared with those obtained by a direct sequencing protocol.RESULTS The LNA-RT-PCR method could identify two rt269L and rt269I polymorphisms of three genotypes,two rt269L types[‘L1’(WT)and‘L2’]and one rt269I type(‘I’)in single(63 samples,72.4%)or mixed forms(24 samples,27.6%)in 87(92.6%sensitivity)of 94 samples from Korean CHB patients.When the results were compared with those obtained by the direct sequencing protocol,the LNA-RT-PCR method showed the same results in all but one of 87 positive detected samples(98.9%specificity).CONCLUSION The newly developed LNA-RT-PCR method could identify two rt269 polymorphisms,rt269L and rt269I,in CHB patients with genotype C2 infections.This method could be effectively used for the understanding of disease progression in genotype C2 endemic areas.

Small Amplicons Mutation Library for Vaccine Screening by Error-Prone Polymerase Chain Reaction

Library construction is a common method used to screen target genes in molecular biology.Most library constructions are not suitable for a small DNA library(<100 base pair(bp))and low RNA library output.To maximize the library’s complexity,error-prone polymerase chain reaction(PCR)was used to increase the base mutation rate.After introducing the DNA fragments into the competent cell,the library complexity could reach 109.Library mutation rate increased exponentially with the dilution and amplification of error-prone PCR.The error-prone PCR conditions were optimized including deoxyribonucleotide triphosphate(dNTP)concentration,Mn^(2+)concentration,Mg^(2+)concentration,PCR cycle number,and primer length.Then,a RNA library with high complexity can be obtained by in vitro transcription to meet most molecular biological screening requirements,and can also be used for mRNA vaccine screening.

Confusing finding of quantitative fluorescent polymerase chain reaction analysis in invasive prenatal genetic diagnosis:A case report

BACKGROUND Quantitative fluorescent polymerase chain reaction(QF-PCR)is a rapid prenatal diagnostic method for abnormalities on chromosomes 21,18,and 13 and sex chromosomal aneuploidy.However,the value of QF-PCR in diagnosing chromosomal structural abnormalities is limited.In this article,we report a confusing QF-PCR finding in a pregnant woman who underwent amniocentesis.CASE SUMMARY The short tandem repeat marker AMXY(Xp22.2/Yp11.2)located on the sex chromosome exhibited a trisomic biallelic pattern,indicating that the karyotype of the fetus might be 47,XYY.Chromosome analysis performed on cultured amniocytes showed a normal male karyotype of the fetus.Copy number variation sequencing confirmed a 500 kb duplication at Yp11.2-Yp11.2(chrY:6610001_7110000)and a 250 kb duplication at Yp11.2-Yp11.2(chrY:7110001_7360000).CONCLUSION In conclusion,the comprehensive application of different methods could achieve a higher detection rate and accuracy for the prenatal diagnosis of chromosomal disorders through chromosomal testing.

Deoxyribonucleic Acid-Polymerase Chain Reaction Status of HIV Exposed Infants in a Sub Regional Prevention of Mother-to-Child Transmission of HIV Programme during the Period 2009-2020

Introduction: Transitioning to more efficacious Antiretrovirals for HIV infected pregnant women and infant prophylaxis has reduced Mother to child transmission of HIV significantly. This study aimed to determine HIV infection status in HIVexposed infants who had their first DNA polymerase chain reaction test in our molecular Laboratory. Subjects, Materials and Methods: Dried Blood Spots for HIV DNA results from 5 states between 2009 and 2020 were analyzed in the PCR laboratory of the Federal Teaching Hospital, Gombe. Results: Nine thousand eight hundred and twenty-three Human Immunodeficiency Virus Deoxyribonucleic acid polymerase Chain Reaction results were analysed;4937 (50.2%) were males. During the study period, there was an overall declining trend in the mother-to-child transmission rate from 3.8% in 2009 to 1.0% in 2020. 6120 (62.3%) of HIV + mothers received Highly active antiretroviral therapy HAART before pregnancy. 7845 (76.2%) of the infants received Nevirapine prophylaxis. Dried blood spot samples were collected from 4077 (41.5%) at 6 - 8 weeks. 8438 (85.9%) received cotrimoxazole. 9469 (96.4%) were ever breastfed. Of the 9823 HIV DNA PCR results, 255 (2.6%) were positive while 69/4077 (1.7%) and 109/2662 (4.1%) were positive for HIV DNA at 6 - 8 weeks and > 12 weeks respectively. (p = 0.001). 86/747 (11.5%) of infants whose HIV-positive mothers received no ARVS were HIV DNA positive. (p = 0.001). 106/884 (12.0%) of infants who had no Antiretroviral prophylaxis had positive HIV DNA results;7/413 (1.7%) with Zidovudine/Nevirapine prophylaxis had positive results. (p = 0.001). 246/9469 (2.6%) of infants that were ever breastfed were positive for HIV DNA;11/354 (3.0%) that never breastfed had positive HIV DNA. Conclusion: Lack of maternal/infant ARVs and prolonged breastfeeding increased the risk of infant HIV infection.

USE OF THE POLYMERASE CHAIN REACTION (PCR) TO DETECT MONOCLONALITY OF B CELL LYMPHO-PROLIFERATIVE DISORDERS

A technique has been developed using PCR to detect monoclonality of B-lymphoproliferative disorders. DNA was extracted from the blood, tissue and paraffin embedded sections by biochemical means or boiling. Forty cases of B-non Hodgkin's lymphoma (NHL), 15 cases of T-NHL, 8 cases of chronic lymphocytic leukemia, 17 cases of reactive lymphadenopathy and 12 cases of various non-lym-phocytic tumor were examined. Monoclonality of B-lymphocytes was detected in 86-92% of cases with B-lymphoproliferative diseases, but none in T-NHL, reactive disorders and non-lymphatic tumors. This technique provides a new molecular biologic method to diagnose malignant B-lymphoproliferative dicor-ders. It may be useful in Ig gene rearrangement study, differential diagnosis and retrospective investigation of lymphoproliferative disorders.

A Molecular Approach to Identification of the Chinese Drug“pu Gong Ying”(Herba Taraxaci)and Six Adulterants byDNA Fingerprinting Using Random Primed PolymeraseChain Resaction(PCR)

DNA fingerprinting among members of the Chinese drug Pu Gong Ying(Taraxacum mongolicum Hand,-Mazz.)and six adulterants of Tu Gong Ying were demonstrated with random-primed polymerase chain reaction(PCR)including arbitrarily primed polymerase chain reaction(AP-PCR)and random amplified polymorphic DNA(RAPD).Distinctive,reproducible genomic fingerprints from DNA from 7 species belonged to Compositae were generated with two long(20 and 24 mer)and one short(10 mer)randomly chosen primers.The Pu Gong Ying can be differentiated from six species of Tu Gong Ying according to the banding pattems of their amplified DNA on agarose gels.The results showed that AP-PCR and RAPD methods can be used for identifying Chinese drugs.Moreover,the Similarity Indexes of the genomic DNA fingerprints showed that Pu Gong Ying and its adulterants are unrelated.Therefore,AP-PCR and RAPD methods can be used for identifying Chinese drugs.

Sequencing of hepatitis C virus cDNA with polymerase chain reaction directed sequencing *

AIM To explore a rapid and easy sequencing method for hepatitis C virus (HCV) genome, and establish a new sequencing method in China. METHODS Polymerase Chain Reaction (PCR) was combined with DNA sequencing technique. PCR products were purified by agarose gel electrophoresis, polyacrylamide gel electrophoresis (PAGE), Polyethylene glycol (PEG) respectively. Then in the presence of a 5′ labeling PCR primer, purified PCR products were directly sequenced. By this method, HCV NS5b cDNA from two HCV infected individuals (HC 42 and HC 49) were sequenced.

3'-terminus shifted bases degeneracy primers increasing sensitivity of polymerase chain reaction

目的:通过使用3’末端碱基游移混合引物,减少引物末端错配,提高多变区基因片段的PCR检测阳性率。方法:在HBV DNA P基因区设计一对检测阳性率相对较高的引物(原型引物),在原型引物序列基础上将两根引物的3’末端分别截去1个或2个碱基,设计出两对参数与原型引物近似的突变引物。分别单独用原型引物和原型引物与突变引物组成的混合引物,在相同条件下对27份HBV DNA阳性标本行PCR,比较二者阳性率。用混合引物扩增4例拉米呋丁治疗无效患者血清HBV DNA,将扩增产物测序并评价3’末端错配对PCR的影响。结果:原型引物和混合引物检测阳性率分别为70.4％(19/27)和85.2％(23/27),两者差异非常显著(P<0.05)。引物3’末端错配能导致PCR假阴性。结论:扩增保守性相对较差的基因片段,采用3’末端单个或多个碱基截短的引物,构成3’末端碱基游移混合引物,可以避免因3’末端错配产生的PCR假阴性,提高检测阳性率。

Study of differential polymerase chain reaction of C-erbB-2 oncogene amplification in gastric cancer

AIM To study the significance of C-erbB-2 oncogene amplification in gastric cancer.METHODS C-erbB-2 oncogene amplification was examined by using differential polymerase chain reaction (dPCR) in surgical and endoscopic specimens of 83 cases of gastric cancer and 101 metastatic lymph nodes.RESULTS C-erbB-2 amplification was found in 28.9% (24/ 83) surgical specimens and 20.5% (17/ 83) endoscopic ones of gastric cancer patients. The amplification was significant in both types of specimens of advanced cancer cases (P＜0.05) and surgical specimens with lymph node metastasis (P＜0.01). The incidence of C-erbB-2 amplification in lymph nodes with metastasis was higher than in primary sites (surgical specimens, P＜0.05). The patients with amplification tumors had poorer 5-year survival rates than those with unamplification ones in the early cancers and well to moderately differentiated adenocarcinomas (P＜0.05). The same surgical samples were tested again by Southern blot hybridization to ascertain C-erbB-2 amplification, and the positive rate of C-erbB-2 amplification (15.7%) was lower than that of dPCR (28.9%, P＜0.05).CONCLUSION Examining C-erbB-2 amplification by dPCR is a quick, simple, reliable and independent method, and is helpful in predicting prognosis and metastatic potential of gastric cancer.

Rapid quantification of semen hepatitis B virus DNA by real-time polymerase chain reaction

AIM： To examine the sensitivity and accuracy of real-time polymerase chain reaction （PCR） for the quantification of hepatitis B virus （HBV） DNA in semen. METHODS： Hepatitis B viral DNA was isolated from HBV carriers＇ semen and sera using phenol extraction method and QIAamp DNA blood mini kit （Qiagen, Germany）. HBV DNA was detected by conventional PCR and quantified by TaqMan technology-based real-time PCR （quantitative polymerase chain reaction （qPCR））. The detection threshold was 200 copies of HBV DNA for conventional PCR and 10 copies of HBV DNA for real time PCR per reaction. RESULTS： Both methods of phenol extraction and QIAamp DNA blood mini kit were suitable for isolating HBV DNA from semen. The value of the detection thresholds was 500 copies of HBV DNA per mL in the semen. The viral loads were 7.5×10^7 and 1.67×10^7 copies of HBV DNA per mL in two HBV infected patients＇ sera, while 2.14×10^5 and 3.02×10^5 copies of HBV DNA per mL in the semen. CONCLUSION： Real-time PCR is a more sensitive and accurate method to detect and quantify HBV DNA in the semen.

Development and application of a real-time polymerase chain reaction method for Campylobacter jejuni detection

AIM:To develop a real-time polymerase chain reaction(PCR) method to detect and quantify Campylobacter jejuni(C.jejuni) from stool specimens.METHODS:Primers and a probe for real-time PCR were designed based on the specific DNA sequence of the hipO gene in C.jejuni.The specificity of the primers and probe were tested against a set of Campylobacter spp.and other enteric pathogens.The optimal PCR conditions were determined by testing a series of conditions with standard a C.jejuni template.The detection limits were obtained using purified DNA from bacterial culture and extracted DNA from the stool specimen.Two hundred and forty-two specimens were analyzed for the presence of C.jejuni by direct bacterial culture and real-time PCR.RESULTS:The optimal PCR system was determined using reference DNA templates,1 × uracil-DNA glycosylase,3.5 mmol/L MgCl 2,1.25 U platinum Taq polymerase,0.4 mmol/L PCR nucleotide mix,0.48 μmol/L of each primer,0.2 μmol/L of probe and 2 μL of DNA template in a final volume of 25 μL.The PCR reaction was carried as follows:95 ℃ for 4 min,followed by 45 cycles of 10 s at 95 ℃ and 30 s at 59 ℃.The detection limit was 4.3 CFU/mL using purified DNA from bacterial culture and 10 3 CFU/g using DNA from stool specimens.Twenty(8.3%,20/242) C.jejuni strains were isolated from bacterial culture,while 41(16.9%,41/242) samples were found to be positive by realtime PCR.DNA sequencing of the PCR product indicated the presence of C.jejuni in the specimen.One mixed infection of C.jejuni and Salmonella was detected in one specimen and the PCR test for this specimen was positive.CONCLUSION:The sensitivity of detection of C.jejuni from stool specimens was much higher using this PCR assay than using the direct culture method.

Detection of bacterial DNA from cholesterol gallstones by nested primers polymerase chain reaction

AIM To search for bacterial DNA sequences in cholesterol gallstones with negative bacterial culture. METHODS DNA was extracted from cholesterol gallstones in gallbladders and nested primers polymerase chain reaction (NP PCR) was used to amplify bacterial gene fragments for identifying the existence of bacteria. The samples of bacterial DNA extracted from potentially causative or unrelated living bacteria were amplified in vitro as the standard markers and comparative 16S ribosomal RNA sequence analysis was made for bacterial identification. RESULTS The gallbladder gallstones of 30 patients were analyzed and bacterial DNA was found in 26 patients. Among them, gallstones with cholesterol content between 30%-69% were seen in 5 (5/5) patients, 70%-90% in 11 (11/14) patients, and more than 90% in 10 (10/11) patients. There was no difference either in cholesterol and water content of gallstones or in harboring bacterial DNA of gallstones. E.coli related DNA fragments appeared in the stones of 8 (26 67%) patients; propionibacteria type DNA in 7 (23 33%); and harbored bacterial gene fragments in 2 patients, similar to Streptococcus pyogenes . A more heterogeneous sequence collection was found in 7 (23 33%) patients, which could belong to multiple bacterial infections. Two (6 67%) patients had bacterial DNA with low molecular weight which might be related to some unidentified bacteria. CONCLUSION Most cholesterol gallstones harbor bacterial DNA. It is important to determine whether these microorganisms are innocent bystanders or active participants in cholesterol gallstone formation.

Rapid detection of sepsis complicating acute necrotizing pancreatitis using polymerase chain reaction

INTRODUCTIONAcute narcotizing pancreatitis usually takes a severe clinical course and is associated with multiple organ dysfunction .With the further understanding of pathophysiological events of acute pancreatisis and the therapeutic measuses taken by the clinicians ,the patients can pass through the critical carry stages ,and then the septic complication caused by rtanslocated bacteria, mostly gram-negative microbes from the intestines ensues[1].

Detection of PERV by polymerase chain reaction and its safety in bioartificial liver support system

AIM： To establish a method detecting porcine endogenous retrovirus （PERV） in China experimental minipigs and to evaluate the safety of PERV in three individuals treated with bioartificial liver support systems based on porcine hepatocytes. METHODS： Porcine hepatocytes were isolated with two-stage perfusion method, then cultured in the bioreactor, which is separated by a semipermeable membrane （0.2μm） from the lumen through which the patients＇ blood plasma was circulated. After posthemoperfusion, patients＇ blood was obtained for screening. Additionally, samples of medium collected from both intraluminal and extraluminal compartments of the laboratory bioreactor and culture supernate in vitro was analyzed. The presence of viral sequences was estimated by polymerase chain reaction （PCR） and reverse transcriptase-polymerase chain reaction （RTPCR）. Finally, the infection of virus in the supernate of common culture was ascertained by exposure to the fetal liver cells. RESULTS： PERV-specific gag sequences were found in the porcine hepatocytes using RT-PCR. and were detected in all samples from the intraluminal, extraluminal samples and culture supernate. However, culture supernatant from primary porcine hepatocytes （cleared of cellular debris） failed to infect human fetal liver cells. Finally, RT-PCR detected no PERV infection was found in the blood samples obtained from three patients at various times post-hemoperfusion. CONCLUSION： The assays used are specific and sensitive, identified by second PCR. PERVs could be released from hepatocytes cultured in bioreactor without the stimulation of mitogen and could not be prevented by the hollow fiber semipermeable membrane, indicating the existence of PERV safety in extracorporeal bioartificial liver support system （EBLSS）.

Comparison of direct fecal smear microscopy,culture,and polymerase chain reaction for the detection of Blastocystis sp.in human stool samples

Objective:To compare the sensitivity and specificity of direct fecal smear microscopy,culture,and polymerase chain reaction in the detection of Blastocystis sp.in human stool.Methods:Human stool samples were collected from a community in San Isidro,Rodriguez,Rizal,Philippines.These samples were subjected to direct fecal smear microscopy,culture and polymerase chain reaction to detect the presence of Blastocystis sp.Results:Of the 110 stool samples collected,28(25%)were detected positive for the presence of Blastocystis sp.by two or more tests.Culture method detected the highest number of Blastocystis-positive stool samples(n=36),followed by PCR of DNA extracted from culture(n=26),PCR of DNA extracted from stool(n=10),and direct fecal smear(n=9).Compared to culture,the sensitivity of the other detection methods were 66.7%for PCR from culture and 19.4%for both PCR from stool and direct fecal smear.Specificity of the methods was high,with PCR from culture and direct fecal smear having97.3%,while PCR from stool at 95.9%.Conclusions:In this study,in vitro culture is the best method for detecting Blastocystis sp.in human stool samples.

Using the polymerase chain reaction coupled with denaturing gradient gel electrophoresis to investigate the association between bacterial translocation and systemic inflammatory response syndrome in predicted acute severe pancreatitis

AIM: To investigate the use of PCR and DGGE to investigate the association between bacterial translocation and systemic inflammatory response syndrome in predicted severe AP.METHODS: Patients with biochemical and clinical evidence of acute pancreatitis and an APACHE Ⅱ score ≥8 were enrolled. PCR and DGGE were employed to detect bacterial translocation in blood samples collected on d1,3, and 8 after the admission. Standard microbial blood cultures were taken when there was clinical evidence of sepsis or when felt to be clinically indicated by the supervising team.RESULTS: Six patients were included. Of all the patients investigated, only one developed septic complications;the others had uneventful illness. Bacteria were detected using PCR in 4 of the 17 collected blood samples. The patient with sepsis was PCR-positive in two samples (taken on d 1 and 3), despite three negative blood cultures. Using DGGE and specific primers, the bacteria in all blood specimens which tested positive for the presence of bacterial DNA were identified as E coli.CONCLUSION: Our study confirmed thatunlike traditional microbiological techniques, PCR can detect the presence of bacteria in the blood of patients with severe AP. Therefore, this latter method in conjunction with DGGE is potentially an extremely useful tool in predicting septic morbidity and evaluating patients with the disease. Further research using increased numbers of patients, in particular those patients with necrosis and sepsis, is required to assess the reliability of PCR and DGGE in the rapid diagnosis of infection in AP.

Hepatitis C virus in human B lymphocytes transformed by Epstein-Barr virus in vitro by in situ reverse transcriptase-polymerase chain reaction

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.

Gastrointestinal tract distribution of Salmonella enteritidis in orally in fected mice with a species-specific fluorescent quantitative polymerase chain reaction

AIM: To identify and understand the regular distribution pattern and primary penetration site for Salmonella enteritidis (S. enteritidis) in the gastrointestinal tract. METHODS: Based on the species-specific DNA sequence of S. enteritidis from GenBank, a species-specific real- time, fluorescence-based quantitative polymerase chain reaction (FQ-PCR) was developed for the detection of S. enteritidis. We used this assay to detect genomic DNA of S. enteritidis in the gastrointestinal tract, including duodenum, jejunum, ileum, cecum, colon, rectum, esophagus and stomach, from mice after oral infection. RESULTS: S. enteritidis was consistently detected in all segments of the gastrointestinal tract. The jejunum and ileum were positive at 8 h post inoculation, and the final organ to show a positive result was the stomach at 18 h post inoculation. The copy number of S. enteritidis DNA in each tissue reached a peak at 24-36 h post inoculation, with the jejunum, ileum and cecum containing high concentrations of S. enteritidis, whereas the duodenum, colon, rectum, stomach and esophagus had low concentrations. S. enteritidis began to decrease and vanished at 2 d post inoculation, but it was still present up to 5 d post inoculation in the jejunum, ileum andcecum, without causing apparent symptoms. By 5 d post inoculation, the cecum had significantly higher numbers of S. enteritidis than any of the other areas (P < 0.01), and this appeared to reflect its function as a repository for S. enteritidis. CONCLUSION: The results provided significant data for clarifying the pathogenic mechanism of S. enteritidis in the gastrointestinal tract, and showed that the jejunum, ileum and cecum are the primary sites of invasion in normal mice after oral infection. This study will help to further understanding of the mechanisms of action of S. enteritidis.