Establishment Of Droplet Digital PCR Detection Method For Group B Streptococcus And The Differentiation Of CD4~+T Cells In Neonates

BackgroundGroup B streptococcus(GBS)is one of main pathogen inducing perinatal infection.These cases can result in pneumonia,bacteremia and meningitis,leading to serious complication.The US CDC(2010)recommends antenatal screening of vaginal/rectal samples for GBS at 35-37 weeks’ gestation.Bacterium culture,the gold standard for the diagnosis of infection,combined with PCR assays can increase the detection rate.Real-time PCR(qRT-PCR)is a rapid test for GBS can possibly overcome this disadvantage of the culture-based screening test as the status of GBS colonization during labor.Nevertheless,it has been limited as it relies on external reference during the detection and is greatly affected by standards and quality controls of experimental procedures.Compared with qRT-PCR technique,droplet digital PCR(ddPCR),a third generation PCR technique developed recently,is able to greatly improve the sensitivity,precision,accuracy and repeatability of detections.Hence,the application of dPCR is suitable to low copy number variation sample and clinical precious specimen detection.GBS infection can induce macrophages to release cytokines and chemokines,and then activate the innate immune response.At the same time,it can induce the cloning and proliferation of the initial CD4+T cells and differentiate into CD4+T cell subsets to participate in the cellular immune response of the body.However the cellular immune characteristics and specific mechanisms of the body are still unclear after the GBS infection of the newborn.Notch signaling plays an important role in the development of T cells and in the differentiation of CD4+T cells.Thus,study the regulation of Notch pathway in GBS infection during the differentiation of Naive CD4+T cells is of great significance to study the defense mechanism of the body after infection of the newborn.Purpose1.Comparison between ddPCR and qRT-PCR in the quantitative detection of GBS.2.Analysis of vaginal perianal secretion in pregnant women by ddPCR.3.Analysis of cytokines leves and the expression of Notch receptor in the umbilical cord blood of the pregnant women infected with GBS.The objective is investigated whether the Notch signaling pathway was involved in differentiation of naive CD4+ T cells after GBS infection.Method1.Comparison of specificity,sensitivity and reproducibility between ddPCR and qRT-PCR in the quantitative detection of GBS.2.Using microbiological culture as the control,ddPCR was used to detect vaginal/anus secretions samples from clinical pregnant women.Evaluation of the application value of this technique in clinical examination.3.Follow up the pregnant women who have confirmed GBS infection.Analysis of the correlation between the copy number of GBS DNA and the severity of clinical symptoms infection.4.Detect the expression difference of Notch receptor in Naive CD4+ T cells of pregnant women with GBS.The result combined with cytokine detection were used to analyze whether the Notch signaling pathway was involved in the differentiation of T cells after GBS infection.Result1.Specificity and accuracy:GBS(COH1 strain)was used as template to verify the specificity of the primers and probe.Both qRT-PCR and droplet digital PCR(ddPCR)showed good linearity in amplification of serially diluted GBS DNA,but ddPCR showed higher sensitivity(detection limit:1 copies/20μL vs.10 copies/20μL)and higher reproducibility(coefficient of variation:1.01-27.45%vs.2.75-47.96%)than qRT-PCR.2.Analysis of vaginal/perianal secretion in pregnant women by ddPCR:the samples included 102 positive cases and 80 negative cases as determined by bacterial culture.The sensitivity and specificity of ddPCR were 98%±1.37%and 92.5±2.94%respectively.In addition,for patients who were positive for GBS infection as determined by bacterial culture,high DNA copy number of GBS(>1000copies/20μL)was correlated with premature rupture of membranes and neonatal infection in the follow-up.3.The levels of cytokines and chemokines in umbilical cord blood infected by GBS:It was found that IFN-y and CXCL10 expression increased in both the asymptomatic and the symptomatic groups compared to the control group(p<0.05);while IL-2 and IL-6 increased only in the symptomatic group(p<0.05).So,IFN-y may be correlated with severity of GBS infection.4.The expression of receptors involved in the Notch signaling pathway were determined by qRT-PCR and western blot.The results showed that in uninfected mothers,the monocytes expressed DLL land Jagged 1,and the naive CD4+ T cells expressed Notchl and Notch2;while in infected mothers,Notchl expression in naive CD4+ T cells significantly increased,suggesting that Notch1 may be involved in the response to GBS infection.ConclutionddPCR showed advantages over qRT-PCR in accuracy and reproducibility,and time consumption is largely reduced compared to bacterial culture,so it is well suited for rapid analysis of clinical samples.And analysis of clinical samples with ddPCR showed that high copy number of GBS in vaginal perianal secretions is correlated with high incidence of premature rupture of membranes and neonatal infection,suggesting ddPCR as a novel method for evaluation of GBS infection during pregnancy.Notch1 expression increased in umbilical cord blood of GBS infected pregnant women,and serum IFN-γ and CXCL10 in umbilical cord blood may be correlated with severity of GBS infection,suggesting that Notchl may be involved in differentiation of naive CD4+ T cells to Thl cells.