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.

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.

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.

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.

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.

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.

BORRELIA BURGDORFERI DNA IN BIOLOGICAL SAMPLES FROM PATIENTS WITH SARCOIDOSIS USING THE POLYMERASE CHAIN REACTION TECHNIQUE

Polymerase chain reaction (PCR) was used to detect the presence of Barrelia burgdoferi DNA in biological samples from patients with sarcoidosis. The target DNA sequence was of chromosomal origin. The amplified DNA sequence was analyzed by agarose gel electrophoresis, PAGE with silver staining, and the identity of amplified DNA was confirmed by restriction enzyme cleavage and DNA-DNA hybridization with a 32P-labelled probe. The assay was sensitive to fewer than two copies of B. burgdorferi genome, even in the presence of a 104-fold excess of human eukaryotic DNA , and was also specific to different B. burgdorferi strains tested. Sera serologically positive to B. burgdcrferi (n=26), bronchoalveolar lavage fluid and supernatant of BALF (n=26) and peripheral blood (n=9) from sarcoidosis patients were tested. The positive rate was low (4/26, 2/26. and 0/9, respectively). It was considered that DNA from B. burgdor ferimay be identified in a minority of patients with sarcoidosis, and it may play a pathogenetic role in such cases. More studies need to be done before advancing the hypothesis of an etiologic role of B. burgdorferi in sarcoidosis.

Quantum dots induce hot-start effects for Taq-based polymerase chain reaction

Decent hot-start effects were here reported in Taq DNA polymerase-based polymerase chain reaction (PCR) when water-soluble CdTe quantum dots (QDs) were employed. The hot-start effects were revealed by the higher amplicon yields and distinguished suppression of nonspecific amplification after pre-incubation of PCR mix with quantum dots between 30°C and 56°C. DNA targets were well amplified even after PCR mixture was pre-incubated 3 hr at 30°C or 1 hr at 50°C. Importantly, the effects of QDs nanoparticles could be reversed by increasing the polymerase concentration, suggesting that there was an interaction between QDs and Taq DNA polymerase. Moreover, control experiment indicated that hot-start effect is not primarily due to the reduced polymerase concentration resulted from the above interaction. This study provided another good start to investigate potential implications of quantum dots in key molecular biology techniques.

Primary application of a real-time quantitative polymerase chain reaction for the detection of human breast cancer related novel gene-Metadherin expression

Objective:The aim of this study was to detect the expression level of Metadherin (MTDH) in peripheral blood of the breast cancer patients by real-time fluorescence quantitative polymerase chain reaction (PCR),and to explore the relationship between expression of Metadherin gene in the patients peripheral blood and the clinic-pathological features in breast cancer. Methods:Real-time fluorescence quantitative polymerase chain reaction was employed to determine the expression level of Metadherin gene in 80 peripheral blood samples of breast cancer patients and healthy donors. Results:The expression of Metadherin gene in breast cancer patients peripheral blood were positive,in which 34 breast cancer patients were highly expressed,accounting for 55.7%,while the expression of Metadherin gene in normal females peripheral blood were negative,there was statistical significance (Ratio = 2.02±0.81,P < 0.05); Ratio of the Metadherin expression in breast cancer patients peripheral blood and the glyceraldehyde-3-phosphate dehydrogenase expression was 1.15 ± 0.36. REST software analysis showed that the expression of Metadherin gene was significantly up-regulated in breast cancer. Conclusion:The SYBR Green I quantitative real-time polymerase chain reaction method can successfully detect the expression level of Metadherin gene. Expression level of Metadherin gene in breast cancer patients peripheral blood is closely related to survival,and it maybe involved in the development of breast cancer and used as an indicator of prognosis.

Sensitivity assay of polymerase chain reaction for detection of Canine Parvo Virus infection in dogs

A polymerase chain reaction was performed using re-ported primers for detection of Canine Parvo virus (CPV) in the stool sample obtained from repository. The PCR primers were specific to VP1/VP2 gene of CPV. Sensi-tivity assay of PCR detection was performed by making dilutions of CPV positive DNA extracted from fecal sample, carrying out PCR for each dilution and visualiz-ing amplicons in ethidium bromide stained agarose gel under UV radiation. Study was valuable in determining the efficiency of PCR. The sensitivity of PCR in present study was determined to be equivalent to detection of .00 2pg/μl of CPV DNA. The study was conducted to analyze the variation, sensitivity and repeatability.

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.

Investigation on detection of Haemophilus ducreyi by Polymerase Chain Reaction

Objective:To investigate the application of polymerase chain reaction (PCR) detection of Haemophilus ducreyi in clinical diagnosis of chancroid. Methods: Nucleotide sequences of 16srRNA gene specific for H. dureyi were used to develop primer sets for amplification of two strains. The amplified products were tested via PCR and sequenced by electrophoresis in a 1.5% gel.These products were compared with those of heterogeneous species or related bacteria to test the specificity of the PCR assay. PCR amplification with different concentrations of H.ducreyi was performed to test its sensitivity. Results: PCR amplification of two strains of H. ducreyi produced a single band of expected 438bp length. The sequence was identified with genomic DNA. None of the other 19 reference species amplified under the same conditions gave this result. The highest sensitivity of PCR assay in the present test was 10ng/L. Conclusions: PCR assay for detection of H. ducreyi is a rapid, specific, and sensitive detection method. If laboratory conditions are strictly controlled, PCR assay is a potentially useful laboratory test for H. ducreyi infection diagnosis.

4种植物源性成分多重real-time PCR检测方法的建立及其在食用淀粉中的应用

建立一种可同时快速检测红薯、木薯、马铃薯、玉米源性成分的多重实时聚合酶链式反应(real-time polymerase chain reaction,real-time PCR)方法。分别以红薯g3pdh基因、木薯g3pdh基因、马铃薯UGPase基因、玉米zSSIIb基因为靶基因设计特异性引物和TaqMan探针,以18S rRNA基因为内参基因,建立多重real-time PCR方法,开展方法学验证,并对不同掺入比例模拟样品和实际淀粉样品进行检测。结果显示,该方法具有高通量、特异性强、灵敏度高等优点。与15种非目标源性均无交叉反应;对目标DNA的检测灵敏度可达到3×10^(-3) ng/μL,且具有良好的线性关系和扩增效率;对淀粉样品的检出限可达0.1%,对50份实际样品进行检测,结果与参比方法一致,说明建立的多重real-time PCR法可用于食用淀粉种类掺假鉴别检测。

Sperm Mediated Transfer of Genes into Goldfish and Detection by Polymerase Chain Reaction

Goldfish sperms were mixed with eggs for fertilization after incubation with antifreeze protein gene(AFP)from ocean pout for 30 min.A number of embryos and 145 adult goldfish were obtained.DNA from adult goldfish and embryos was extracted separately.Results of the amplification by PCRand Southern blot molecular hybridization indicate the integration of exogenous antifreeze gene intothe genome of a part of the recipient goldfish.Of the 45 samples detected by PCR,twelve showedpositive reaction with distinct hybridization band.The positive rate was 26%.

Development and Clinical Application of a Single-tube Nested PCR Method to Amplify the DNA Polymerase Ⅰ Gene of Treponema Pallidum

Objective: To develop a sensitive, specific and simple method for detection of extremely low numbers of T. pallidum in clinical specimens, as a significant addition to the serologic tests for syphilis diagnosis. Methods: Double-tube nested PCR(DN-PCR) and single-tube nested PCR(SN-PCR) assays were performed to amplify specific fragments of the DNA poly-merase I gene(polA) of T. pallidum. Sensitivity and specificity of the two PCR assays were tested. Eighty-six whole blood specimens from persons with suspected syphilis were detected by the two nested PCR methods. The TPPA test was used as a comparison for detecting syphilis in sera from corresponding patients. Results: Only specific amplicons could be obtained during amplification of the T. pallidum polA gene and the detection limit was approximately 1 organism when analyzed on gel by the two PCR methods. Of 86 clinical specimens, 62 were positive by TPPA. Of these, 54 and 51 were positive by the DN-PCR and SN-PCR, respectively, which does not represent a statistically significant difference between the two PCR tests. Of 24 TPPA-negative specimens, 5 were positive by both DN-PCR assay and SN-PCR assay. Conclusion: The SN- polA PCR method is extremely sensitive, specific and easy to perform for detecting low numbers of T. pallidum in clinical blood specimens as a complementary to serology for syphilis diagnosis.

基于主成分分析的多重定量PCR荧光串扰校正

聚合酶链式反应(PCR)是分子生物学常用的检测手段,主要用于对生物的DNA或RNA进行检测。由于荧光光谱重叠和滤光片过滤带宽限制,检测时所获得的荧光数据通常会包含荧光通道之间的串扰,串扰的存在使PCR结果分析变得复杂,并可能影响最终的检测结果。选择合适的光学元件,并确定通道间的补偿矩阵,可以降低甚至消除荧光串扰。目前荧光补偿矩阵大多通过迭代计算获得,还没有一种简单的方法可以从混合的多通道荧光数据中找到荧光补偿矩阵。为了快速获得荧光补偿矩阵,减小计算量,采用主成分分析法(PCA)中确定主成分的方式,基于搭建的测试平台进行单一染料实验,获得染料的荧光信号在各个检测通道的分布情况,计算得到荧光补偿矩阵。通过分析补偿矩阵,发现对于搭建的硬件系统,Cy5染料对Cy5.5通道串扰较大,串扰比例为8.76%,同时Cy5.5染料对Cy5通道串扰影响也相对较大,比例约为6.2%;其次是ROX染料对HEX通道串扰,比例约为2.68%;HEX染料对FAM通道串扰,比例约为1.58%;FAM染料对HEX通道串扰相对较小,比例约为0.25%,其余通道无明显串扰,与荧光光谱反映的结果一致。采用得到的荧光补偿矩阵对单一染料实验得到的原始荧光数据进行处理,有效去除了非目标通道的荧光串扰,实现了荧光通道数据的解耦,验证了方法的可行性。最后设计了染料颜色分辨实验,将不同浓度的多种染料进行组合测试,并采用所提出的方法将得到的数据进行荧光补偿。实验结果表明,荧光通道各自的线性相关性较高,五个荧光通道的线性相关系数r均大于0.99,该结果进一步验证了该补偿方法的有效性。

多重PCR毛细管电泳细菌快速鉴定方法的建立和临床应用研究

目的针对引起人类感染的常见病原菌建立一种基于多重PCR毛细管电泳技术的快速细菌鉴定方法,评估其临床应用价值。方法建立23种常见病原菌的多重PCR毛细管电泳检测体系。收集150例临床微生物检测标本,分别用多重PCR毛细管电泳法(以下简称多重PCR法)、培养法进行检测。对两种方法的检测结果进行比较,评价多重PCR法的检测效能。结果150例标本中多重PCR法检出15种病原菌、培养法检出14种病原菌。多重PCR法检测阳性率为73.3%,培养法为70.0%,差异无统计学意义(P>0.05)。多重PCR法对肺炎链球菌的检出率为16.0%,培养法为6.0%,差异有统计学意义(P<0.05)。多重PCR法对混合菌标本的检出率为15.3%,培养法未检出混合菌标本。多重PCR法与培养法检测结果的符合率为79.3%。多重PCR法检测时间为3~6 h,培养法为2~4 d。结论与培养法比较,多重PCR法具有较高的时效性,对肺炎链球菌及混合菌标本具有较高的检出率,可满足临床标本病原微生物快速初筛的需求。

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.