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.

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.

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.

Clinical Value of the Fluorescent Quantitative Polymerase Chain Reaction in the Diagnosis of Primary Syphilis

Objective： To evaluate the clinical value of fluorescent quantitative polymerase chain reaction （FQ-PCR） in the diagnosis of primary syphilis. Methods： 68 swab specimens were collected from patients suspected of infecttion with primary syphilis attending two STD clinics （Guangzhou Institute of Dermatovenerology and the First People＇s Hospital of Guangzhou city）, from September 1998 to December 2000. Analysis： by FQ-PCR, darkfield microscopy （D-F） for Treponema pallidum （TP）, and serologic testing for syphilis （STS）. Results： Of 68 patients, 30 （44.12%） were positive for TP by QF-PCR assay, 19 （27.94%） were positive for TP by D-F, 33 （48.53%） were positive for TP-IgG antibody by RPR, and 42 （61.76%） were positive for TP-IgG antibody by TPHA. There are significant differences in detection between D-F and TPHA （P〈0.05）, but there is no difference with RPR （P〉0.1）. Conclusion： This data shows that QF-PCR is a convenient, reliable and rapid method for diagnosis of primary syphilis, and may be an effective clinical assay in the detection of TP.

Two－step multiplex polymerase chain reaction for gene diagnosis of progressive pseudohypertrophic muscular dystrophy

in the present study,9 exon-containing DNA segments of dystrophin gene with 9 sets of oligonucleotide primers by two-step multiplex polymerase chain reaction (mPCR) were amplified. Subsequently,gene analysis was performed in 36 cases of Duchenne mascular dystroply (DMD) and 4 cases of Becker muscular dystrophy(BMD). The findings showed that 17 cases of deletion were detected by using the first 5 sets of primers with a relatively high incidence of deletion detection and 2 more cases of deletion were detected by using the remaining 4 sets of primers. The total deletion rate detected by mPCR with 9 cases of primers was 47. 5% of the patients examined,suggesting that about 79. 1% of the patients with gene deletion could be detected. Thus,as a preliminary screening, the two-step mPCR can be used in the gene diagnosis of DMD/BMD. The method is not only simple, convenient and rapid,but also free from radiosotope trouble.

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].

Use of polymerase chain reaction on maternal peripheral blood for determination of fetal sex

On the basis of the characteristics of Y-chromosome sequence which consists of Y specific repeat DNA family(DYZI) of 800￣5 000 copies,a pair of primers Y3,Y4 is designed to amplify the specific DNA segment of 446 bp by the polymerase chain reaction(PCR),so as to detect the presence of Y chromosome in male fetal cell in maternal peripheral blood for the purpose of prenatal determination of fetal sex.The authors made use of PCR amplification of crude DNA from maternal peripheral blood in early, mid,and late pregnancies to determine the fetal sex.Comparison of the results with those of PCR of their corresponding chorionic villi and amniotic fluid,and with the sex of the aborted fetus or the new born showed coincident rates of 93%,100%,and 87. 5% in early, mid,and late pregnancies respectively.The procedure offers a new alternative way for non-invasive prenatal diagnosis,

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.

Carrier Screening and Prenatal Gene Diagnosis of β-thalassemia by PCR-RDB Technique

In order to identify the distribution of gene types of β-thalassemia and reduce the birthrates of β-thalassemia major in Guiyang area, 1054 pregnant women and their spouses from Affiliated Hospital, Guiyang Medical College were screened. The positive samples were analyzed with polymerase chain reaction and reverse dot blot method (PCR-RDB). When both partners were heterozygous identified as carriers for β- thalassemia, the risk of having a fetus who was homozygous or compound heterozygous was 2.66 %; the ratio of male to female was 1/1.15. Seven types of mutation were identified. CD17 and CD41-42 were dominant among them. Among the 4 cases subject to prenatal gene diagnosis, one fetus was completely normal and 3 fetuses were diagnosed as having β-thalassemia major (1 homozygous and 2 compound heterozygous). The fetuses diagnosed as β-thalassemia major were selectively terminated within two weeks. It was concluded that the birthrate of β-thalassemia major in Guiyang area was reduced and the target of improving birth outcome and child development has been achieved.

miRNA表达谱在产前诊断胎儿先天性心脏病中的研究

目的:探究微小RNA(microRNA,miRNA)表达谱在产前诊断胎儿先天性心脏病(congenital heart disease,CHD)中的应用。方法:收集2021年1月—2022年12月于天津市中心妇产科医院就诊的30例超声确诊为CHD的孕妇(病例组)和同期10例要求行羊水穿刺的孕妇(对照组),用Illumina测序平台对2组孕妇的羊水上清进行全转录组测序,2组孕妇的全部miRNA进行归一化,分析差异表达的miRNA。从差异表达的miRNA中挑选P<0.05和|log2 FC|>3(差异倍数,Fold Change,FC)的miRNA再在羊水和外周血中进行实时荧光定量聚合酶链反应(real time fluorescence quantitative polymerase chain reaction,RT-qPCR)验证,比较羊水中miRNA测序与RT-qPCR的差异倍数,挑选外周血与羊水表达调控方向一致的miRNA。结果:共发现138个差异表达miRNA,其中85个上调,53个下调。进一步挑选出了15个差异表达的miRNA,羊水中miRNA测序与RT-qPCR结果比较相一致。外周血与羊水中表达调控方向一致的miRNA有2个,分别为miR-222-3p和miR-189-5p,这2个miRNA在病例组母血中表达量较对照组显著上升(均P<0.05)。结论:母血中miRNA作为新的血清学标志物可以初步应用于筛查胎儿CHD。

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.

鹦鹉喙羽症病毒PCR检测方法的建立与应用

为建立一种灵敏特异的检测鹦鹉喙羽症病毒PCR方法,给临床快速诊断鹦鹉喙羽症提供技术支持,根据Genbank上公开的鹦鹉喙羽症病毒基因序列,比对分析找出高保守区域,设计了1对检测引物,提取鹦鹉喙羽症病毒阳性样品DNA模板,对扩增体系和反应条件进行一系列调整,确定理想的PCR反应方案;通过10倍梯度稀释DNA模板法,确定该检测方法的灵敏度极限;应用该方法检测传染性法氏囊炎病毒、鸡传染性支气管炎病毒、新城疫病毒、传染性喉气管炎病毒、鸡痘病毒和Ⅰ群禽腺病毒4型这6种禽病病原,确定方法的特异性;运用该PCR方法检测90份临床样品,测试其实用性。结果表明,设计的检测引物能特异性扩增鹦鹉喙羽症病毒,其它6种家禽病毒均为阴性,其检测DNA浓度的下限是3.725×10^(-6)ng/μL。临床应用表明,90份疑似病料有28份为阳性,阳性率为31.1%。成功建立了一种灵敏度高、特异性好的鹦鹉喙羽症病毒PCR方法。

数字聚合酶链反应技术对甲状腺结节术前良恶性的鉴别诊断价值

目的探讨数字聚合酶链反应(dPCR)技术在甲状腺结节术前良恶性鉴别诊断中的价值。方法收集2019年6月至2022年9月在该院进行超声引导下细针穿刺细胞学检查(FNAC)且接受手术治疗的甲状腺结节患者穿刺液标本141份,采用dPCR和扩增阻滞突变系统(ARMS)-PCR检测BRAF V600E基因突变,dPCR同时检测NRAS Q61R基因突变、TERT基因启动子C228T和C250T突变。以患者术后病理检测结果为金标准,比较几种方法的准确率,评价dPCR技术在甲状腺结节术前良恶性鉴别诊断中的价值。结果141份穿刺液标本中,dPCR对BRAF V600E的检出率为69.50%(98/141),ARMS-PCR对BRAF V600E的检出率为64.54%(91/141),差异有统计学意义(P<0.05);dPCR检测NRAS Q61R、TERT C228T、TERT C250T在甲状腺乳头状癌(PTC)中的突变检出率分别为15.32%(17/111)、12.61%(14/111)和1.80%(2/111)。单独使用dPCR检测BRAF V600E鉴别诊断甲状腺结节良恶性的准确率为89.36%,明显高于ARMS-PCR(84.40%)和FNAC(77.30%),差异均有统计学意义(P<0.05)。dPCR(BRAF V600E)+FNAC的诊断准确率为97.16%,dPCR多基因[BRAF V600E+NRAS Q61R+TERT(C228T+C250T)]+FNAC的诊断准确率为97.87%,均高于单独dPCR(BRAF V600E)、dPCR(多基因)的诊断准确率,差异均有统计学意义(P<0.05)。结论dPCR可检测出ARMS-PCR漏检的基因突变位点,也可以弥补FNAC的不足,该技术联合FNAC可提高甲状腺结节术前良恶性鉴别诊断的效能。

布鲁氏菌病实验室诊断研究进展

布鲁氏菌病是一种具有多种临床表现的人畜共患病,由于临床表现多样且缺乏特异性症状和体征,因此实验室诊断对该疾病的确诊、治疗至关重要。布鲁氏菌的传统实验室检测可分为培养、鉴定、血清学试验。核酸检测法是近年来新兴起的检测手段,可快速准确诊断布鲁氏菌病并对布鲁氏菌进行分型,多种分子诊断技术已在科研领域应用于布鲁氏菌的检测。本文将对目前国内外布鲁氏菌实验室检测方法的最新进展及应用进行阐述并比较其优缺点,为布鲁氏菌病的诊断及治疗提供更全面的指导。

实时荧光PCR检测在支原体肺炎诊断中的应用价值分析

目的探讨在支原体肺炎诊断中选择实时荧光聚合酶链式反应(Polymerase Chain Reaction,PCR)检测的价值,并分析该方案的诊断效能。方法选取2022年8月—2023年10月来宾市人民医院收治的1200例疑似支原体肺炎患儿为研究对象,所有患儿均采集血清和呼吸道分泌物,用化学发光法测定血清中肺炎支原体抗体,用实时荧光PCR法检测呼吸道分泌物中的肺炎支原体核酸(Mycoplasma Pneumoniae Nucleic Acid,MP-DNA),以病原学检查结果为金标准,对比各个方案的诊断效能,分析不同年龄段患儿的肺炎支原体DNA检测结果。结果实时荧光PCR检测灵敏度(99.36%)、特异度(98.08%)、准确度(99.08%)、阳性预测值(99.36%)、阴性预测值(98.08%)高于MP血清学方法检测,差异有统计学意义(χ^(2)=16.312、7.686、23.788、7.595、16.119,P均<0.05)。结论在支原体肺炎诊断中应用实时荧光PCR检测方案可提高准确率,同时该方案具有早期、快速、简便等优势,可为临床医师制定治疗方案提供参考。

Natural history of cytomegalovirus infection in a series of patients diagnosed with moderate-severe ulcerative colitis

AIM： To evaluate the natural history of human cytomegalovirus （HCMV） infection in a series of 28 ulcerative colitis patients in whom the search for HCMV was positive. METHODS： A series of 85 patients with moderate-se- vere ulcerative colitis flare-up were evaluated for a HCMV search by performing a haematoxylin and eosin stain, immunohistochemical assay and nested polymerase chain reaction on rectal biopsies. Among 85 screened patients （19 of whom were steroid resistant/dependant）, 28 were positive for HCMV; after remission the patients were followed up clinically and histologically. RESULTS： Among the 22 patients with complete follow- up, in 8 （36%） patients HCMV-DNA persisted in the in- testinal specimens. Among the HCMV positive patients, 4 （50%） experienced at least one moderate-severeflare-up of colitis without evidence of peripheral HCMV. Among the 14 HCHV negative patients, 3 with pouches developed pouchiUs and 5 out of 11 （45%） experienced a colitis flare-up. CONCLUSION： Our preliminary results suggest that HCHV may remain in the colon afber an acute coltis flare- up despite remission; it seems that the virus is not responsible for the disease relapse.

Clinical significance of cytomegalovirus infection in patients with inflammatory bowel disease

Cytomegalovirus(CMV) infection is common in humans.The virus then enters a "latency phase" and can reactivate to different stimuli such as immunosuppression.The clinical significance of CMV infection in inflammatory bowel disease is different in Crohn's disease(CD) and ulcerative colitis(UC).CMV does not interfere in the clinical course of CD.However,CMV reactivation is frequent in severe or steroid-resistant UC.It is not known whether the virus exacerbates the disease or simply appears as a bystander of a severe disease.Different methods are used to diagnose CMV colitis.Diagnosis is classically based on histopathological identification of viral-infected cells or CMV antigens in biopsied tissues using haematoxylin-eosin or immunohistochemistry,other tests on blood or tissue samples are currently being investigated.Polymerase chain reaction performed in colonic mucosa has a high sensitivity and a positive result could be associated with a worse prognosis disease;further studies are needed to determine the most appropriate strategy with positive CMV-DNA in colonic mucosa.Specific endoscopic features have not been described in active UC and CMV infection.CMV colitis is usually treated with ganciclovir for several weeks,there are different opinions about whether or not to stop immunosuppressive therapy.Other antiviral drugs may be used.Multicenter controlled studies would needed to determine which subgroup of UC patients would benefit from early antiviral treatment.

Cytomegalovirus frequency in neonatal intrahepatic cholestasis determined by serology, histology, immunohistochemistry and PCR

AIM: To determine cytomegalovirus (CMV) frequency in neonatal intrahepatic cholestasis by serology, histological revision (searching for cytomegalic cells), immunohistochemistry, and polymerase chain reaction (PCR), and to verify the relationships among these methods.METHODS: The study comprised 101 non-consecutive infants submitted for hepatic biopsy between March 1982 and December 2005. Serological results were obtained from the patient's f iles and the other methods were performed on paraffin-embedded liver samples from hepatic biopsies. The following statistical measures were calculated: frequency, sensibility, specif ic positive predictive value, negative predictive value, and accuracy.RESULTS: The frequencies of positive results were as follows: serology, 7/64 (11%); histological revision, 0/84; immunohistochemistry, 1/44 (2%), and PCR, 6/77 (8%). Only one patient had positive immunohistochemical fi ndings and a positive PCR. The following statistical measures were calculated between PCR and serology: sensitivity, 33.3%; specificity, 88.89%; positive predictive value, 28.57%; negative predictive value, 90.91%; and accuracy, 82.35%. CONCLUSION: The frequency of positive CMV varied among the tests. Serology presented the highest positive frequency. When compared to PCR, the sensitivity and positive predictive value of serology were low.

Clinical,radiological and molecular diagnosis correlation in serum samples from patients with osteoarticular tuberculosis

Objective:To assess the role of polymerase chain reaction(PCR)in serum sauples,in the diagnosis of osteoarticular tuberculosis(OTB)in a setting where only clinical and imaging diagnoses determine the treatment.Methods:A total of 44 consecutive serum specimens were collected from clinically suspected OTB patients,based on clinical and radiological[X-ray or magnetic resonance imagng/computecl tomography]features.They were scrcened by in-house nested PCR.In addition,a few specimens were examined by Gram stain,acid-fast bacilli stain,histand routine bacterial culture.A total of 39 specimens were collected from patients suffering from other bone diseases of nontuberculous origin and included as negative controls.Results:of the 44 clinically suspected OTB patients,in-house nested PCR was positive in 40(91%)cases;PCR was negative in 38(97%)negative controls.Sensitivity and specificity of our in—house nested PCR was 90.3%and 97.4%,respectively.The PCR report was available within 48 h.It was possible to standardize serum PCR technique and in positive cases,a good n was observed in terms of an adequate treatment response.Conclusions:Nested PCR in serum samples is a rapid,highly sensitive and specific modality for OTB detection,PCR should be performed in addition to clinical evaluation,imaging studies,acidfast bacilli staining,culture and histopathology diagnosis,if possible.