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 majority 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 signifi- cantly 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 early 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 prog- nostic significance.

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.

Quantitative studies of the regular distribution pattern for Salmonella enteritidis in the internal organs of mice after oral challenge by a specific real-time polymerase chain reaction

AIM:To identify and understand the regular distribution pattern for Salmonella enteritidis (S. enteritidis) in the internal organs of mice after an oral challenge over a 3 wk period. METHODS:Assays based on the serovar-specific DNA sequence of S. enteritidis from GenBank, and a serovar-specific real-time, fluorescence-based quantitative polymerase chain reaction (FQ-PCR) were developed for the detection of S. enteritidis. We used this assay to detect genomic DNA of S. enteritidis in the blood and the internal organs, including heart, liver, spleen, kidney, pancreas, and gallbladder, from mice after oral challenge at different time points respectively.RESULTS:The results showed that the spleen was positive at 12 h post inoculation (PI), and the blood was at 14 h PI. The organism was detected in the liver and heart at 16 h PI, the pancreas was positive at 20 h PI, and the final organs to show positive results were the kidney and gallbladder at 22 h PI. The copy number of S. enteritidis DNA in each tissue reached a peak at 24-36 h PI, with the liver and spleen containing high concentrations of S. enteritidis, whereas the blood, heart, kidney, pancreas, and gallbladder had low concentrations. S. enteritidis populations began to decrease and were not detectable at 3 d PI, but were still present up to 12 d PI in the gallbladder, 2 wk for the liver, and 3 wk for the spleen without causing apparent symptoms. CONCLUSION:The results provided significant data for understanding the life cycle of S. enteritidis in the internal organs, and showed that the liver and spleen may be the primary sites for setting itself up as a commensa over a long time after oral challenge. Interestingly, it may be the first time reported that the gallbladder is a site of carriage for S. enteritidis over a 12 d period. This study will help to understand the mechanisms of action of S. enteritidis infection in vivo.

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.

Rapid genotyping of human rotavirus using SYBR green real-time reverse transcription-polymerase chain reaction with melting curve analysis

AIM: To develop a real-time reverse transcriptionpolymerase chain reaction(RT-PCR) assay to genotype rotavirus(G and P) in Alberta from January 2012 to June 2013. METHODS: We developed and validated a different approach to perform rotavirus G and P genotyping using a two-step SYBR green RT-PCR(rt-g PCR) by selecting genotype-specific primers of published conventional RT nested PCR(cn RT-PCR) assay and optimizing the amplification conditions. c DNA was first synthesized from total RNA with Super Script? Ⅱ reverse transcriptase kit followed by amplication step using monoplex SYBR green real-time PCR. After the PCR reaction, melting curve analysis was used to determine specific genotype. Sixteen samples previously genotyped using cn RT-PCR were tested using the new assay and the genotyping results were compared as sensitivity analysis. Assay specificity was evaluated by testing other gastroenteritis viruses with the new assay. The amplicon size of each available genotype was determined by gelelectrophoresis and DNA sequences were obtained using Sanger-sequencing method. After validation and optimization, the new assay was used to genotype 122 pediatric clinical stool samples previously tested positive for rotavirus using electron microscopy between January2012 and June 2013.RESULTS: The new rt-g PCR assay was validated and optimized. The assay detected G1 to G4, G9, G12 and P[4] and P[8] that were available as positive controls in our laboratory. A single and clear peak of melting curve was generated for each of specific G and P genotypes with a Tm ranging from 80 ℃ to 82 ℃. The sensitivity of rt-g PCR was comparable to cn RT-PCR with 100% correlation of the 16 samples with known G and P genotypes. No cross reaction was found with other gastroenteritis viruses. Using the new rt-g PCR assay, genotypes were obtained for 121 of the 122 pediatric clinical samples tested positive for rotavirus: G1P[8](42.6%), G2P[4](4.9%), G3P[8](10.7%), G9P[8](10.7%), G9P[4](6.6%), G12P[8](23.0%), and unknown GP[8](0.8%). For the first time, G12 rotavirus strains were found in Alberta and G12 was the second most common genotype during the study period. Gel electrophoresis of all the genotypes showed expected amplicon size for each genotype. The sequence data of the two G12 samples along with other genotypes were blasted in NCBI BLAST or analyzed with Rota C Genotyping tool(http://rotac.regatools.be/). All genotyping results were confirmed to be correct.CONCLUSION: rt-g PCR is a useful tool for the genotyping and characterization of rotavirus. Monitoring of rotavirus genotypes is important for the identification of emerging strains and ongoing evaluation of rotavirus vaccination programs.

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。

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.

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). Methods Based on the gene-specific region of the following pathogens: Chlamydia trachomatis omp1/ompb, herpes simplex virus (HSV) DNA polymerase, Treponema pallidum 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.

Identifying the stability of housekeeping genes to be used for the quantitative real-time PCR normalization in retinal tissue of streptozotocin-induced diabetic rats

AIM:To investigate the stability of the seven housekeeping genes:beta-actin(ActB),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),18s ribosomal unit 5(18s),cyclophilin A(CycA),hypoxanthine-guanine phosphoribosyl transferase(HPRT),ribosomal protein large P0(36B4)and terminal uridylyl transferase 1(U6)in the diabetic retinal tissue of rat model.METHODS:The expression of these seven genes in rat retinal tissues was determined using real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR)in two groups;normal control rats and streptozotocininduced diabetic rats.The stability analysis of gene expression was investigated using geNorm,NormFinder,BestKeeper,and comparative delta-Ct(ΔCt)algorithms.RESULTS:The 36B4 gene was stably expressed in the retinal tissues of normal control animals;however,it was less stable in diabetic retinas.The 18s gene was expressed consistently in both normal control and diabetic rats’retinal tissue.That this gene was the best reference for data normalisation in RT-qPCR studies that used the retinal tissue of streptozotocin-induced diabetic rats.Furthermore,there was no ideal gene stably expressed for use in all experimental settings.CONCLUSION:Identifying relevant genes is a need for achieving RT-qPCR validity and reliability and must be appropriately achieved based on a specific experimental setting.

Real-time Fluorescence PCR Method for Detection of Burkholderia glumae from Rice

Burkholderia glumae causing seedling rot and grain rot of rice was listed as a plant quarantine disease of China in 2007. It＇s quite necessary to set up effective detection methods for the pathogen to manage further dispersal of this disease. The present study combined the real-time PCR method with classical PCR to increase the detecting efficiency, and to develop an accurate, rapid and sensitive method to detect the pathogen in the seed quarantine for effective management of the disease. The results showed that all the tested strains of B. glumae produced about 139 bp specific fragments by the real-time PCR and the general PCR methods, while others showed negative PCR result. The bacteria could be detected at the concentrations of 1×10^4 CFU/mL by general PCR method and at the concentrations below 100 CFU/mL by real-time fluorescence PCR method. B. glumae could be detected when the inoculated and healthy seeds were mixed with a proportion of 1：100.

4种动物源性成分多重real-time PCR检测方法的建立及其在驴肉制品检测中的应用

建立一种同时快速检测驴、马、猪及鸭源性成分的四重实时聚合酶链式反应(real-time polymerase chain reaction,real-time PCR)方法。分别以4种源性成分的Nad5、ATpase6、ATP8、cytb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,并对该方法进行方法学验证,同时对不同掺入比例模拟样品、不同加工工艺模拟样品和实际驴肉样品进行检测。结果显示,该方法具有高通量、特异性强、灵敏度高等优点。当Ct值≤35.0时,方法对16种非目标源性具有良好特异性;灵敏度可检测到质量浓度为2×10^(-4)ng/μL的模板DNA;对生肉的检出限为肉含量的0.001%,对熟肉制品的检出限为肉含量的0.01%;对100份实际样品进行检测,结果与标准方法一致,说明建立的多重real-time PCR法可用于肉及肉制品中常见掺假源性成分的检测。

3种致病菌多重real-time PCR检测方法的建立及其在散装即食肉制品中的应用

建立一种同时快速检测沙门氏菌、金黄色葡萄球菌和蜡样芽孢杆菌的三重实时聚合酶链式反应(realtime polymerase chain reaction,real-time PCR)方法。以沙门氏菌invA基因、金黄色葡萄球菌Sa442基因和蜡样芽孢杆菌Cereolysin AB基因为靶基因设计引物和TaqMan探针,建立多重real-time PCR方法,对该方法进行方法学验证,并对实际的散装即食肉制品样品进行检测。结果表明,该方法特异性强、灵敏度高、重复性好。对15株非目标菌进行检测,结果均为阴性;3种致病菌的定量线性浓度范围为10^(2)~10^(8) CFU/mL,且定量检测批内和批间的变异系数均小于2%;沙门氏菌、金黄色葡萄球菌和蜡样芽孢杆菌在未增菌的即食肉制品样品中检出限分别为3.8×10^(2)、4.9×10^(2) CFU/mL和5.7×10^(2) CFU/mL,经增菌5 h后,检出限提高到3.8、4.9 CFU/mL和5.7 CFU/mL。对100份实际样品进行检测,结果与标准方法一致,说明建立的多重real-time PCR法可以用于散装即食肉制品中3种致病菌的检测。

三重real-time PCR检测副溶血弧菌主要毒力基因

针对编码副溶血弧菌的特异性基因和主要毒力基因tlh、tdh、ureR进行引物及探针设计,通过优化反应条件,建立基于Taqman探针快速检测副溶血弧菌毒力基因的三重实时聚合酶链式反应(real-time polymerase chain reaction,real-time PCR)方法。利用10株副溶血弧菌和22株非副溶血弧菌对设计的引物及探针进行特异性验证,结果表明设计的引物及探针具有很高的特异性;优化后的引物浓度分别为tdh 0.15μmol/L、tlh 0.15μmol/L、ureR 0.80μmol/L,探针浓度分别为HEX 0.50μmol/L、FAM 0.50μmol/L;该方法即使在高浓度的背景细菌存在下,DNA浓度与Ct值均呈良好的线性关系,检出限为1.8×10^(2)拷贝/mL;以完整菌细胞的悬液为模板时,预变性时间为30 min,扩增检测效果与以基因组DNA(gDNA)为模板相当(ΔCt<1)。本研究建立的三重real-time PCR方法能实现快速定量检测副溶血弧菌,并能有效区分致病性及非致病性副溶血弧菌,为副溶血弧菌的快速定量检测和风险评估提供快速、灵敏、准确的方法。

Use of real-time polymerase chain reaction for the diagnosis of Pneumocystis pneumonia in immunocompromised patients： a meta-analysis

Background The diagnosis of Pneumocystis pneumonia （PCP） in immunocompromised patients is still challenging today due to the absence of an in vitro culture system and the low diagnostic accuracy of microscopic examinations. Herein, we performed a meta-analysis to evaluate the accuracy of real-time polymerase chain reaction （PCR） in the diagnosis of PCP. Methods We searched Web of Knowledge and Medline from 1990 to May 2010 for studies reporting diagnostic accuracy data regarding the use of real-time PCR in the diagnosis of PCP in immunocompromised patients. Results Ten individual studies were included. Overall, the sensitivity of real-time PCR was 97% （95% CI： 93%-99%）; the specificity was 94% （95% CI： 90%-96%）. The area under the HSROC curve （95% CO for real-time PCR was 0.99 （0.97-0.99）. In a subgroup analysis regarding studies involving HIV patients among the study population, the sensitivity and specificity were 97% （95% CI： 93%-99%） and 93% （95% CI： 89%-96%）, respectively. Regarding studies using Bronchoalveolar lavage （BAL） samples only： sensitivity =98% （95% CI： 94%-99%）; specificity =93% （95% CI： 89%- 96%）, respectively. Regarding studies using microscopy as a reference standard： sensitivity =97% （95% CI： 92%-99%）; specificity =93% （95% CI： 88%-96%）. However, high between-study statistical heterogeneity was observed in all analyses. Conclusions Real-time PCR has a good diagnostic accuracy and may provide a useful adjunctive tool for the diagnosis of PCP in immunocompromised patients. Further studies are needed in order to identify any differences in the diagnostic performance of real-time PCR in HIV and non-HIV immunocompromised patients.

Micro-droplet Digital Polymerase Chain Reaction and Real-Time Quantitative Polymerase Chain Reaction Technologies Provide Highly Sensitive and Accurate Detection of Zika Virus

The establishment of highly sensitive diagnostic methods is critical in the early diagnosis and control of Zika virus(ZIKV)and in preventing serious neurological complications of ZIKV infection. In this study, we established micro-droplet digital polymerase chain reaction(ddPCR) and real-time quantitative PCR(RT-qPCR) protocols for the detection of ZIKV based on the amplification of the NS5 gene. For the ZIKV standard plasmid, the RT-qPCR results showed that the cycle threshold(Ct) value was linear from 10~1 to 10~8 copy/l L, with a standard curve R^2 of 0.999 and amplification efficiency of 92.203%;however, a concentration as low as 1 copy/l L could not be detected. In comparison with RT-qPCR, the dd PCR method resulted in a linear range of 10~1–10~4 copy/l L and was able to detect concentrations as low as 1 copy/l L. Thus, for detecting ZIKV from clinical samples, RT-qPCR is a better choice for high-concentration samples(above 10~1 copy/l L),while ddPCR has excellent accuracy and sensitivity for low-concentration samples. These results indicate that the ddPCR method should be of considerable use in the early diagnosis, laboratory study, and monitoring of ZIKV.

A rapid and sensitive method to detect mycobacterium tuberculosis DNA by fluorescence polarization

Objective: To develop a new high sensitivity, rapid and simple mycobacterium tuberculosis DNA detection method using fluorescence polarization technology. Methods: In our asymmetric PCR protocol, 100 times mole of TB-R primer was added than in the usual symmetric PCR to get enough single strands PCR product. The probe TB-5′-TAMRA and PCR products were mixed in a tube and incubate for 5-15 min at 46 ℃.The polarization (mp) was measured using victor2 Multilabel Plate Reader. Results: Asymmetric and symmetric PCR products were analyzed by the FP method. Asymmetric PCR products are detected more sensitively than symmetric ones. The polarization values of probe associated with asymmetric products were much higher than with symmetric products. Conclusion: This fluorescence polarization assay in conjunction with asymmetric PCR is a powerful and widely applicable method for the rapid and sensitive detection of micro-organisms in clinical laboratories.