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.

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.

Detection of hepatitis C virus in serum and peripheral blood mononuclear cells by two reverse transcription polymerase chain reactions

Over 75% of patients with acute hepatitis C （HCV） will develop into chronic infection associated with chronic hepatitis, end stage liver disease or hepatocellular carcinoma. HCV infection is usually ignored because the level of HCV in serum of some patients is very low and most patients with chronic HCV infection are asymptomatic. Some patients with persistent abnormal liver function may be infected with HCV although HCV RNA and HCV antibodies in serum are negative because their HCV load is under the current detectable limit or sometimes HCV is absent in serum but present in the liver or peripheral blood mononuclear cells （PBMCs） and other tissues.

Polymerase chain reaction-based tests for detecting Helicobacter pylori clarithromycin resistance in stool samples: A meta-analysis

BACKGROUND Helicobacter pylori(H.pylori)infection is closely associated with the etiology of a variety of gastric diseases.The effective eradication of H.pylori infection has been shown to reduce the incidence of gastric carcinoma.However,the rate of H.pylori eradication has significantly declined due to its increasing resistance to antibiotics,especially to clarithromycin.Therefore,the detection of clarithromycin resistance is necessary prior to the treatment of H.pylori.Although many studies have been conducted on the use of polymerase chain reaction(PCR)-based tests to detect clarithromycin resistance in stool samples,no accurate data on the feasibility of these tests are available.Here,we performed a meta-analysis to assess the feasibility of these noninvasive tests.AIM To evaluate the reliability of PCR-based tests for detecting H.pylori clarithromycin resistance in stool samples.METHODS We searched PubMed,Medline,Embase,and other databases for articles that evaluated the value of the PCR analysis of stool samples for detecting the resistance of H.pylori to clarithromycin.We collected cross-sectional studies that met the inclusion criteria.Diagnostic accuracy measures were pooled using a random-effects model.The risk of bias was assessed using the Quality Assessment of Diagnostic Accuracy Studies-2 tool.Subgroup analysis was also conducted according to PCR type,purification technique,reference standard,mutation site,sample weight,number of patients,and age group,and the clinical utility of diagnostic tests was evaluated using the Likelihood Ratio Scatter Graph.RESULTS Out of the 1818 identified studies,only 11 met the eligibility criteria,with a total of 592 patients assessed.A meta-analysis of the random-effect model showed that PCR-based analysis of stool samples had high diagnostic accuracy for detecting clarithromycin resistance in patients infected with H.pylori.The combined sensitivity was 0.91[95%confidence interval(CI):0.83-0.95],Q=30.34,and I2=67.04,and the combined specificity was 0.97(95%CI:0.62-1.00),Q=279.54,and I2=96.42.The likelihood ratio for a positive test was 33.25(95%CI:1.69-652.77),and that for a negative test was 0.10(95%CI:0.05-0.18),with an area under the curve of 0.94.The diagnostic odds ratio was 347.68(95%CI:17.29-6991.26).There was significant statistical heterogeneity,and the sub-analyses showed significant differences in the number of patients,sample weight,purification methods,PCR types,mutation points,and reference standards.The included studies showed no risk of publication bias.CONCLUSION PCR-based tests on stool samples have high diagnostic accuracy for detecting H.pylori clarithromycin resistance.

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.

Expression of cellular fibronectin mRNA in adult periodontitis and peri-implantitis: a real-time polymerase chain reaction study

Cellular fibronectin （cFn） is a type of bioactive non-collagen glycoprotein regarded as the main substance used to maintain periodontal attachment. The content of cFn in some specific sites can reflect the progress of periodontitis or peri-implantitis. This study aims to evaluate the expression of cFn messenger RNA （mRNA） in tissues of adult periodontitis and peri-implantitis by real-time fluorescent quantitative polymerase chain reaction （PCR） and to determine its clinical significance. A total of 30 patients were divided into three groups of 10： healthy, adult periodontitis and peri-implantitis. Periodontal tissue biopsies （1 mmx I mmx I mm） from each patient were frozen in liquid nitrogen. Total RNA was extracted from these tissues, and the content, purity and integrity were detected. Specific primers were designed according to the sequence, and the mRNA expression levels of cellular fibronectin were detected by real-time PCR. The purity and integrity of the extracted total RNA were both high, and the specificity of amplified genes was very high with no other pollution. The mRNA expression of cFn in the adult periodontitis group （1.526＋0.441） was lower than that in the healthy group （3.253＋0.736）. However, the mRNA expression of cFn in the peri-implantitis group （3.965＋0.537） was significantly higher than that in the healthy group. The difference revealed that although both processes were destructive inflammatory reactions in the periodontium, the pathomechanisms were different and the variation started from the transcription level of the cFn gene.

Real-time polymerase chain reaction for the diagnosis of necrotizing herpes stromal keratitis

AIM： To design, optimize and validate a rapid,internally controlled real-time polymerase chain reaction（RT-PCR） test for herpes simplex virus（HSV） in the diagnosis of necrotizing herpes stromal keratitis.· METHODS： Tears alone or together with corneal epithelium scrapings from 30 patients（30 eyes）suspected of necrotizing herpes stromal keratitis were tested for HSV DNA by RT-PCR. The samples were collected during the first visit and then on the subsequent 7, 14, 28, 42, and 56 d. The symptoms of the patients were scored before treatment to determine the correlation between HSV concentration in the corneal epithelium scrapings and clinical scores.·RESULTS： The positive rate（46.4%） in the corneal epithelium group before the therapy was significantly higher than that（13.3%） in the tears group（P =0.006）.There were 13 positive HSV patients before the therapy,the concentration of HSV DNA in corneal epithelium scrapings group was significantly higher than that in the tears group（paired t-test, P =0.0397）. Multilevel mixedeffects model analysis showed that the difference between the corneal epithelium scrapings group and the tears group was statistically significant（P =0.0049）. The Spearman rank correlation analysis indicated a positive correlation between the HSV concentration in the corneal epithelium scrapings and clinical scores before the treatment（r =0.844, P〈 0.0001）.· CONCLUSION： RT-PCR appears to be a powerful molecular tool for the diagnosis of necrotizing herpes stromal keratitis.

An improved electrochemiluminescence polymerase chain reaction method for the detection of Fusarium wilts

An improved electrochemiluminescence polymerase chain reaction （ECL-PCR） method was developed and applied to detect Fusarium wilt. Briefly, the internal transcribed spacer （ITS） sequence of Fusarium oxysporumf, sp Cubense （FOC） was amplified by PCR. Two universal fragments, which were complimentary to Ru（bpy）3^2＋ （TBR） labeled probe and Biotin labeled probe, respectively, were connected to the tail of primers so that all the PCR products got universal sequences. Then biotin labeled probes and TBR labeled probes were hybridized with the PCR products at the same time. Through the specific interaction between biotin and streptavidin, the PCR products were captured by streptavidin coated magnetic bead and then detected by ECL assay. The experiment results showed that the healthy banana samples and infected ones can be discriminated by this ECL-PCR method. This improved ECL-PCR approach is useful in Fusarium wilt detection due to its high sensitivity, simplicity and stability.

Development of Mutiplex Polymerase Chain Reaction for the Detection and Differentiation of Enteric Adenoviruses in Stool Samples

To find a technique of detecting and differentiating enteric adenoviruses (EAds) in clinical samples, a novel PCR approach was developed. EAds were able to be detected by use of a pair of subgroup F general primers (P1 and P2), and they were also be able to be differentiated from each other in the presence of another adenovirus type 40 (Ad40) specific primer (P3) in the same tube. Our results showed that there was one band for Ad41 and two bands for Ad40, respectively, on running-gel after PCR performance. PCR was performed on 40 specimens in parallel directly with dot-hybridization assay on the same diluted stool samples. 20 of 40 specimens were positive by hybridization (of them 12 were Ad41 and 8 were Ad40), whereas 26 were positive by PCR performance on the same samples with Ad41 18 and Ad40 8 positive as well. Our study indicated that this novel method could be used in clinical laboratory or in epidemic investigation for Eads

Droplet digital polymerase chain reaction assay for methylated ring finger protein 180 in gastric cancer

BACKGROUND Gastric cancer(GC)is one of the most prevalent malignant tumors that endangers human health.Early diagnosis is essential for improving the prognosis and survival rate of GC patients.Ring finger protein 180(RNF180)is involved in the regulation of cell differentiation,proliferation,apoptosis,and tumorigenesis,and aberrant hypermethylation of CpG islands in the promoter is strongly associated with the occurrence and development of GC.Thus,methylated RNF180 can be used as a potential biomarker for GC diagnosis.AIM To use droplet digital polymerase chain reaction(ddPCR)to quantify the methylation level of the RN180 gene.A reproducible ddPCR assay to detect methylated RNF180 from trace DNA was designed and optimized.METHODS The primer and probe were designed and selected,the conversion time of bisulfite was optimized,the ddPCR system was adjusted by primer concentration,amplification temperature and amplification cycles,and the detection limit of ddPCR was determined.RESULTS The best conversion time for blood DNA was 2 h 10 min,and that for plasma DNA was 2 h 10 min and 2 h 30 min.The results of ddPCR were better when the amplification temperature was 56°C and the number of amplification cycles was 50.Primer concentrations showed little effect on the assay outcome.Therefore,the primer concentration could be adjusted according to the reaction system and DNA input.The assay required at least 0.1 ng of input DNA.CONCLUSION In summary,a ddPCR assay was established to detect methylated RNF180,which is expected to be a new diagnostic biomarker for GC.

A quantitative polymerase chain reaction assay for the enumeration of brown tide algae Aureococcus anophagefferens in coastal waters of Qinhuangdao

Aureococcus anophagefferens, a small pelagophyte algae, has caused brown tide blooms in coastal waters of Qinhuangdao in recent years, presenting significant negative impacts on the shellfish mariculture industry. Under standard light microscopy, it is visually indistinguishable from other small algae in field samples due to its extremely small size. In this study, quantitative polymerase chain reaction（q PCR） based on 18 S r DNA sequences was developed and used to detect and enumerate A. anophagefferens. A linear regression（R2 = 0.91） was generated based on cycle thresholds value（Ct） versus known concentrations of A. anophagefferens. Twenty-two field samples collected in coastal waters of Qinhuangdao were subjected to DNA extraction and then analyzed using q PCR. Results showed that A. anophagefferens had a wide distribution in coastal waters along Qinhuangdao. Elevated A. anophagefferens abundance, category 3 brown tide blooms（〉200 000 cells/m L） occurred at Dongshan Beach and Tiger-stone Beach in August in 2013. In shellfish mariculture areas along coastal waters of Qinhuangdao, 4 stations had category 3 blooms, and 6 stations had category 2 blooms（35 000–200 000 cells/m L） in August and all stations had category 1 blooms（〉0 to ≤35 000 cells/m L） in October. Quantitative PCR allows for detection of A. anophagefferens cells at low levels in filed samples, which is essential to effective management and prediction of brown tide blooms.

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.

Quantification of Porcine Follicle-stimulating Hormone Receptor Messenger Ribonucleic Acid by Reverse Transcription competitive Polymerase Chain Reaction

An easy and reliable method was developed for construction and quantification of competitive templates, which shared the same sequence as the amplified target DNA except for a 20 bp insertion in the middle by recombinant polymerase chain reaction (PCR). Among the advantages of competitive PCR is that any predictable or unpredictable variable that affects amplification has the same effect on both target and competitor species and that the final ratio of amplified products reflects exactly the initial targets. The utilization of a thermostable reverse transcriptase in the RT step was proposed to overcome the problem of the efficiency of target cDNA synthesis. In addition, to obtain reliable measurements, it was recommended to perform four PCR with amounts of competitive template flanking the concentration of the target mRNA.

Detection of Human Papillomavirus DNA in Oral Cancer Tissue Using Polymerase Chain Reaction

Using polymerase chain reaction (PCR), 25 specimens from 22 patients with oral carcinomas were examined by 6 selected primers of human papillomavirus (HPV). Eighteen of the 22 patients (18/22) gave positive reaction with a positive rate of 81.8%. The positive rates of HPV 6, 11, 16 and 18 were 27.3%, 18.2%, 63.6% and 40.9% respectively. 13.6% positive for mixed infection of HPV 16 and 18 (3/22) and 18.2% positive for mixed infection of HPV, 6, 11, 16 and 18 (4/22). Examining enlarged cervical lymph nodes in three cases with suspecting metastases to cervical lymph nodes from oral carcinomas. It revealed HPV DNA 16 and 18 in two cases and HPV DNA 18 in one case. These results suggested that there was a tendency for HPV 16 and 18 to metastasinze via lymphatics. Only one case of the three had a pathologic diagnosis of lymph node metastasis. Of the 30 non tumor controls, HPV DNA positivity was 10%, all being HPV 18. χ 2 test gave a P<0.005. It strongly indicated that HPV 16 and 18 were related to oral carcinomas.

Detection and Identification of Enteroviruses RNA by Using Polymerase Chain Reaction

For rapid diagnosis of enteroviral infection in clinic practice, we developed a reverse transcription and polymerase chain reaction (RT-PCR) assay.Primers homologous to the conserved 5' non-coding region were designed by analyzing enteroviral genomes, and then they were used to enzymatically amplify RNA from 31 prototype enteroviral strains and enteroviruses (EV) in cerebrospinal fluid (CSF) of 34 cases of aseptic meningitis and 11 cases of aseptic encephalitis. The RT-PCR products generated with these enteroviral primers were analyzed by agar gel electrophoresis and dot blot hybridization analysis. 31 EV strains showed an obvious monoclonal amplification band, and all dot blot hybridization results were positive. Four other viruses and cells cultured were all negative. The study of sensitivity of the RT-PCR showed that amplification production were positive to 10(-2)- 10(-3) 50% tissue culture infective doses. With this assay, 21(61.8%) of 34 aseptic meningitis and 8 (72. 7%) of 11 aseptic encephalitis contained EV RNA in CSF samples. Two cases of meningitis and one of encephalitis with EV infection were still positive during convalescence. Our results suggest that this RT-PCR method was a fast, sensitive and specific technique for detection of common EV infection.

Detection of Gene Alteration for Color Vision Defects by Polymerase Chain Reaction

According to the fact that the abnormalities of visual pigment genes were always involved in the changing of the exon 5, two oligonucleotide primers were designed to amplify the exon 5 of red pigment gene and green pigment gene. After electrophoresis of the PCR products digested with Rsal or Sau3A, the DNA fragments from the exon 5 of red pigment gene (RPG) and green pigment gene (GPG) were separated since there are different restriction endonuclease sites. On the other hand, we analyzed the exon 5 rela...