Quick recovery and characterization of cell-free DNA in seminal plasma of normozoospermia and azoospermia： implications for non-invasive genetic utilities

We established a quick and reliable method for recovering cell-free seminal DNA （cfsDNA）, by using the binding-washing-elution procedure on the DNA purification column. Low variations （below 15%） among the triplicate values of cfsDNA quantity verified the reproducibility of our cfsDNA recovery method. Similar cfsDNA yield and size distribution between seminal plasma acquired by filtration and centrifugation confirmed the presence of cfsDNA. To investigate the general characterization of cfsDNA, the quantitation and size distribution of cfsDNA from normozoospermic and azoospermic semen were analyzed by real-time PCR and electrophoresis, respectively. CfsDNA concentration in semen with normozoospermia （n = 11） was 1.34 ± 0.65 μg ·mL^-1, whereas a higher cfsDNA concentration was observed in azoospermia （2.56 ± 1.43 μg ·mL^-1, n = 9）. The continuous distribution of DNA fragments ranging from -1 kb to 15 kb and a spectrum of multiples of 180-bp fragments were observed in each normozoospermic and azoospermic sample. Distinct characteristic DNA ladder fragmentations in some azoospermic samples implicated that cfsDNA originate partly from apoptotic cells. CfsDNAs of 36 selected azoospermic patients with known information of Y chromosome microdeletion were subjected to the same microdeletion analysis by multiplex PCR and PCR amplification of sY114 （1 450 bp）. All multiplex PCR reactions with cfsDNA amplified successfully and provided the same result as leukocyte DNA. PCR amplification of sY114 gave a 1 450-bp amplicon as expected. Our data suggested the potential use of cfsDNA in search of biomarker or diagnostic procedures.

Detecting plasma Epstein-Barr virus DNA to diagnose postradiation nasopharyngeal skull base lesions in nasopharyngeal carcinoma patients:a prospective study

The diagnosis of postradiation nasopharyngeal skull base lesions in petients with nasopharyngeal carcinoma(NPC) is still a tough problem in clinical practice.An early and accurate diagnosis is important for subsequent management.We prospectively evaluated the diagnostic value of plasma Epstein-Barr virus(EBV) DNA in detecting postradiation nasopharyngeal skull base lesions in NPC patients.From July 2006 to September 2010,90 patients with postradiation NPC(34 women and 56 men;median age:42 years) met the selection criteria and were recruited in this study.All postradiation nasopharyngeal skull base lesions were found in the latest magnetic resonance imaging(MRI) examinations before endoscopic surgery,and the nasopharyngeal cavity was normal under flexible nasopharyngoscopy.Plasma EBV DNA detection was performed within 2 weeks before endoscopic surgery.A total of 90 endoscopic operations were successfully performed without any postoperative complications.Recurrences confirmed by postoperative pathology were found in 30 patients.The specificity,positive and negative predictive values of plasma EBV DNA detection were better than those of MRI.In addition,combining plasma EBV DNA detection with MRI improved the specificity and positive predictive values of MRI.Plasma EBV DNA detection followed by MRI would help to diagnose recurrence whereas MRI was unable.These results indicate that plasma EBV DNA is an effective and feasible biomarker for detecting postradiation nasopharyngeal skull base lesions in NPC patients.

The Effect of Atmospheric Pressure Plasma Corona Discharge on pH, Lipid Content and DNA of Bacterial Cells

Effect of plasma corona discharges on the pH, whole cell lipids and DNA of bacteria is investigated. Results showed an increase in the acidity levels of water due to plasma reactive species which, however, were not responsible for bacterial cell death. No changes in the whole cell lipid contents were observed, while DNA after plasma treatment showed deterioration of the amplified sequences, indicating the possible occurrence of DNA degradation. In conclusion, reactive species produced by plasma discharges affects DNA, possibly contributing to cell death.

Fabrication of an electrochemical sensor for determination of doxorubicin in human plasma and its interaction with DNA

In this work, an electrochemical sensor was fabricated for determination of an anthracycline, doxorubicin(DOX) as a chemotherapy drug in plasma based on multi-walled carbon nanotubes modified platinum electrode(Pt/MWCNTs). DOX was effectively accumulated on the surface of modified electrode and generated a pair of redox peaks at around 0.522 and 0.647 V(vs. Ag/Ag Cl) in Britton Robinson(B-R) buffer(p H 4.0, 0.1 M). The electrochemical parameters including p H, type of buffer, accumulation time, amount of modifier and scan rate were optimized. Under the optimized conditions, there was a linear correlation between cathodic peak current and concentration of DOX in the range of 0.05–4.0 μg/m L with the detection limit of 0.002 μg/m L. The number of electron transfers(n) and electron transfer-coefficient(α) were estimated as 2.0 and 0.25, respectively. The constructed sensor displayed excellent precision, sensitivity, repeatability and selectivity in the determination of DOX in plasma. Moreover, cyclic voltammetry studies of DOX in the presence of DNA showed an intercalation mechanism with binding constant(K_b) of 1.12×10~5L/mol.

Prognostic value of plasma Epstein-Barr virus DNA level during posttreatment follow-up in the patients with nasopharyngeal carcinoma having undergone intensity-modulated radiotherapy

Background: The value of Epstein-Barr virus(EBV) DNA assay during posttreatment follow-up of the patients with nasopharyngeal carcinoma(NPC) presenting with different pretreatment plasma EBV DNA levels remains unclear. In the present study, we aimed to evaluate the prognostic value of plasma EBV DNA assay during posttreatment followup in the patients with NPC who have undergone intensity-modulated radiotherapy.Methods: The medical records of 385 NPC patients treated with intensity-modulated radiotherapy between November 2009 and February 2012 were reviewed. All patients underwent plasma EBV DNA assays before treatment, within3 months after treatment, and then every 3-12 months during posttreatment follow-up period. The recurrence rates for patients with different pretreatment and posttreatment follow-up plasma EBV DNA levels were analyzed.Results: Of the 385 patients, 267(69.4%) had detectable pretreatment plasma EBV DNA(> 0 copy/mL) and 93(24.2%) had detectable posttreatment EBV DNA during a median follow-up of 52.8 months(range 9.3-73.8 months).Detectable EBV DNA during posttreatment follow-up was found in 14.4%(17/118) and 28.5%(76/267) of patients with undetectable and detectable pretreatment EBV DNA, respectively, and was significantly associated with tumor recurrence in both patient groups. EBV DNA was detectable in 12.8%(40/313) of patients who remained disease-free,56.4%(22/39) of patients with locoregional recurrence alone, and 93.9%(31/33) of patients with distant metastasis as the first recurrence event(P < 0.001); 6.5%(19/292) of patients with undetectable EBV DNA and 57.0%(53/93) of patient with detectable EBV DNA during posttreatment follow-up experienced tumor recurrence. Compared with other cut-off values, the cut-off value of 0 copy/mL for EBV DNA during posttreatment follow-up had the highest area under the ROC curve(AUC) value(0.804,95% confidence interval 0.741-0.868) for predicting tumor recurrence(sensitivity, specificity, and accuracy: 73.6%, 87.2%, and 84.7%, respectively).Conclusion: Plasma EBV DNA level during posttreatment follow-up is a good marker for predicting distant metastasis but not locoregional recurrence in the patients with NPC irrespective of the pretreatment EBV DNA levels.

Gene Analysis of Free Fetal DNA in Maternal Plasma

To investigate the feasibility of using free fetal DNA from maternal plasma as the source of fetal material in non invasive prenatal diagnosis, SRY gene of free DNA in maternal blood of 65 samples were analyzed by using primer extension preamplication (PEP) and probe microplate hybridization techniques. The results showed that the detection rate of SRY gene in maternal blood from women carrying male fetuses detected by probe microplate hybridization alone and probe microplate hybridization with PEP were 76.09 % (35/46) and 95.65 % (44/46) respectively, and there was a significant difference between them. The non detection rate of SRY gene in blood samples from women carrying female fetus was 100 % (19/19). It is indicated that probe microplate hybridization was an effective method in detecting trace fetal DNA from maternal plasma and the sensitivity could be substantially improved by combined use of the two techniques. Analysis of fetal DNA in maternal plasma can serve as an alternative for non invasive prenatal diagnosis.

基于中医证候与精液质量相关参数构建精子DNA碎片预测模型与验证

背景:中医证候与精液质量相关参数相结合,共同预测精子DNA碎片指数(DNA fragmentation index,DFI)异常增高的发生并绘制列线图,能显著提高临床的实操性与应用效能,为临床全面评估精液质量,采取积极干预措施以改善临床结局及制定个体化医疗方案提供依据。目的:探讨基于中医证候与精液质量相关参数构建精子DNA碎片的预测模型与验证。方法:回顾性分析2019年7月至2021年7月在广西壮族自治区南溪山医院中医男科接受中医证候诊断及精子DNA碎片率检查的不育患者共420例,据《人类精液检查与处理实验室手册》(第6版),将其中137例精子DFI>30%患者纳入精子DFI异常增高组,将283例精子DFI≤30%作为对照组;首先采用单因素分析筛选精子DFI异常增高的影响因素,然后采用套索算法(LASSO)校正因子共线性问题并筛选出最佳匹配因子后,将其纳入多因素向前逐步Logistic回归找出其独立影响因素并绘制列线图,最后采用受试者工作曲线、校准曲线、临床决策曲线、临床影响曲线对该预测模型进行区分度与准确度及临床应用效能验证。结果与结论:①单因素分析结果显示,年龄、体质量指数、前向运动率、精子总活率、精子浓度、精子形态学、肾阳虚衰证、湿热下注证、肾精不足证为引发精子DFI异常增高的影响因子(P<0.05);②通过LASSO回归进一步筛选出的最佳匹配因素为年龄、体质量指数、精子总活率、精子浓度、精子形态学、肾阳虚衰证、湿热下注证、肾精不足证(P<0.05);③多因素向前逐步Logistic回归结果显示年龄、体质量指数、精子浓度、精子总活率、湿热下注证、肾阳虚衰证共6项为引发精子DFI异常增高的独立影响因素;④受试者工作曲线显示,模型组曲线下面积为0.760(0.713,0.806),验证组曲线下面积为0.745(0.714,0.776),说明该预测模型具有较好的区分度;⑤校准曲线平均绝对误差0.040,Hosmer-Lemeshow检验P>0.05,表明该模型预测发生精子DFI异常增高的概率与实际发生精子DFI异常增高的概率无显著统计学差异,证实该模型具有较好的准确度;⑥临床决策曲线与临床影响曲线显示,模型组与验证组分别在阈概率值为0.08-0.84与0.09-0.78时具有临床最大净获益,且在该阈概率范围内具有较好的临床应用效能;⑦结果表明,年龄、体质量指数、精子浓度、精子总活率、湿热下注证、肾阳虚衰证为引发精子DFI异常增高的独立影响因素,通过其构建的临床预测模型列线图具有较好的临床预测价值与临床应用效能,可为临床全面评估精液质量、预后与干预及个体化医疗服务提供依据。

Value of dynamic plasma cell-free DNA monitoring in septic shock syndrome: A case report

BACKGROUND Mortality due to septic shock is relatively high.The dynamic monitoring of plasma cell-free DNA(cfDNA)can guide the treatment of septic shock.CASE SUMMARY Herein,we present a typical case of septic shock syndrome caused by the bacilli Acinetobacter baumannii and Pantoea.The patient complained of abdominal pain,fever and chills upon admission to the Emergency Department.Marked decreases in white blood cells and procalcitonin(PCT)were observed after the patient received continuous renal replacement and extracorporeal membrane oxygenation.Plasma cfDNA levels were consistently high,peaking at 1366.40 ng/mL,as measured by a duplex real-time PCR assay with an internal control,which was developed as a novel method for the accurate quantification of cfDNA.The patient died of septic shock on HD 8,suggesting that cfDNA could be used to monitor disease progression more effectively than PCT and the other inflammatory factors measured in this case.CONCLUSION CfDNA may be a promising marker that complements other inflammatory factors to monitor disease progression in patients with septic shock.

Non-invasive Prenatal Gene Diagnosis: Progress through Cell-free Fetal DNA and RNA in Maternal Plasma and Urine

Non-invasive prenatal gene diagnosis has been developed rapidly in the recent years, and numerous medical researchers are focusing on it. Such techniques could not only achieve prenatal diagnosis accurately, but also prevent tangential illness in fetuses and thus, reduce the incidence of diseases. Moreover, it is non-invasive prenatal gene diagnosis that prevents potential threaten and danger to both mothers and fetuses. Therefore, it is welcomed by clinical gynecologist and obstetrian, researchers of medical genetics, and especially, pregnancies. This review article touches briefly on the advanced development of using cell-free DNA, RNA in maternal plasma and urine for non-invasive prenatal gene diagnosis.

Quantitative analysis of plasma HBV DNA for early evaluation of the response to transcatheter arterial embolization for HBV-related hepatocellular carcinoma

AIM： To assesse changes in plasma HBV DNA after TAE in HBV-related HCC and correlate the levels with the pattern of lipiodol accumulation on CT. METHODS： Between April and June 2001, 14 patients with HBV-associated HCC who underwent TAE for inoperable or recurrent tumor were studied. Levels of plasma HBV DNA were measured by real-time quantitative PCR daily for five consecutive days after TAE. More than twofold elevation of circulating HBV DNA was considered as a definite elevation. Abdominal CT was performed 1-2 mo after TAE for the measurement of lipiodol retention. RESULTS： Circulating HBV DNA in 10 out of 13 patients was elevated after TAE, except for one patient whose plasma HBV DNA was undetectable before and after TAE. In group Ⅰ patients （n = 6）, the HBV DNA elevation persisted for more than 2 d, while in group Ⅱ （n = 7）, the HBV DNA elevation only appeared for i d or did not reach a definite elevation. There were no significant differences in age or tumor size between the two groups. Patients in group Ⅰ had significantly better lipiodol retention （79.31±28.79%） on subsequent abdominal CT than group Ⅱ （18.43±10.61%） （P = 0.02）. CONCLUSION： Patients with durable HBV DNA elevation for more than 2 d correlated with good lipiodol retention measured 1 mo later, while others associated with poor lipiodol retention. Thus, circulating HBV DNA may be an early indicator of the success or failure of TAE.

Codon 249 mutation in exon 7 of p53 gene in plasma DNA:maybe a new early diagnostic marker of hepatocellular carcinoma in Qidong risk area,China

AIM: One of the characteristics of hepatocellular carcinoma (HCC) in Qidong area is the selective mutation resulting in a serine substitution at codon 249 of the p53 gene (1,20),and it has been identified as a 'hotspot' mutation in heptocellular carcinomas occurring in populations exposed to aflatoxin and with high prevalence of hepatitis B virus carriers (2, 3, 9, 10, 16, 24). We evaluated in this paper whether this 'hotspot' mutation could be detected in cellfree DNA circulating in plasma of patients with hepatocellular carcinoma and cirrhosis in Qidong, China, and tried to illustrate the significance of the detection of this molecular biomarker.METHODS: We collected blood samples from 25hepatocellular carcinoma patients, 20 cirrhotic patients and 30 healthy controls in Qidong area. DNA was extracted and purified from 200 μl of plasma from each sample. The 249ser p53 mutation was detected by restriction digestion analysis and direct sequencing of exon-7 PCR products.RESULTS: We found in exon 7 of p53 gene G→T transversion at the third base of codon 249 resulting 249Arg→249ser mutation in 10/25 (40%) hepatocellular carcinoma cases,4/20 (20%) cirrhotics, and 2/30 (7 %) healthy controls.The adjusted odds ratio for having the mutation was 22.1(95 % CI, 3.2～91.7) for HCC cases compared to controls.CONCLUSION: These data show that the 249ser p53mutation in plasma is strongly associated with hepatocellular carcinoma in Qidong patients. We found this mutation was also detected, although it was at a much lower frequency,in plasma DNA of Qidong cirrhotics and healthy controls;We consider that these findings, together with the usual method of HCC diagnosis, will give more information in early diagnosis of HCC, and 249ser p53 mutation should be developed to a new early diagnostic marker for HCC.

Application of Fetal DNA in Maternal Plasma in Noninvasive Prenatal Diagnosis

To explore the application of fetal DNA in maternal plasma for noninvasive prenatal diagnosis, the DNA template was extracted by hydroxybenzene chloroform from 44 maternal (7-41 weeks) plasma. The Fetus derived Y sequence DYZ 1 gene (149bp) was chosen to be amplified by PCR. The fragment was identified in all the plasma of male bearing pregnant women with the diagnostic accordance rate being 100.00 %. Two of the 22 female bearing pregnant women had false positive results. Among the 44 pregnant women, the diagnostic accordance rate was 88.89 % at early pregnant stage, 100.00 % at medium pregnant stage, and 96.55 % at late stage respectively. The final accuracy of 95.45 % was obtained in all cases. It was concluded that by means of hydroxybenzene chloroform extraction the authors of this article promoted the concentration and purity of the DNA template, and diagnosed more accurately. The results showed that free fetal DNA in the maternal plasma could be regarded as the gene resource for noninvasive prenatal diagnosis.

Multiple z-Score Based Method for Noninvasive Prenatal Test Using Cell-Free DNA in Maternal Plasma

Objective: To improve the detecting accuracy of chromosomal aneuploidy of fetus by non-invasive prenatal testing (NIPT) using next generation sequencing data of pregnant women’s cell-free DNA. Methods: We proposed the multi-Z method which uses 21 z-scores for each autosomal chromosome to detect aneuploidy of the chromosome, while the conventional NIPT method uses only one z-score. To do this, mapped read numbers of a certain chromosome were normalized by those of the other 21 chromosomes. Average and standard deviation (SD), which are used for calculating z-score of each sample, were obtained with normalized values between all autosomal chromosomes of control samples. In this way, multiple z-scores can be calculated for 21 autosomal chromosomes except oneself. Results: Multi-Z method showed 100% sensitivity and specificity for 187 samples sequenced to 3 M reads while the conventional NIPT method showed 95.1% specificity. Similarly, for 216 samples sequenced to 1 M reads, Multi-Z method showed 100% sensitivity and 95.6% specificity and the conventional NIPT method showed a result of 75.1% specificity. Conclusion: Multi-Z method showed higher accuracy and robust results than the conventional method even at low coverage reads.

新型超声快速处理活检标本保存不同年限对DNA质量的影响

背景:新型超声组织处理技术越来越多地被用来进行分子生物学分析,研究新型超声处理不同存储年限组织DNA的质量,对进一步分子检测的标本质控具有重要意义。目的:探讨新型超声处理活检标本存储不同年限对DNA质量的影响,以期为分子检测探索最佳的标本存储时间。方法:收集40例乳腺穿刺小活检组织,采用超声技术制作石蜡标本,按照存储年限分为4组:<1年组、1-3年组、>3-5年组及>5年组,每组10例,对石蜡标本进行切片,每张切片厚3μm,切片10-15张,提取DNA后通过Nanophotometer N60超微量分光光度计和Qubit 4.0荧光计检测DNA的质量浓度,记录A_(260)/A_(280)比值判定DNA的纯度,利用全自动毛细管电泳核酸分析仪(Qsep 100)检测DNA片段完整性,以评估DNA片段的质量。结果与结论:4组样本A_(260)/A_(280)均值在1.8-2.0之间,达到纯度要求,无明显差异。4组样本的DNA质量浓度(Qubit浓度)均值分别为30.39,14.33,2.52,1.95 ng/μL;DNA的平均N/Q比值分别为6.48,14.18,24.56,29.86;DNA质量数均值分别为5.64,1.76,1.24,0.80;大片段占比均值分别为56.08%,17.72%,12.68%,7.90%。PCR检测内控基因Ct均值分别为15.32,17.09,18.39,21.24。与<1年组相比,其余3组DNA浓度显著降低,N/Q比值显著增加,DNA质量数和大片段占比均值显著降低,Ct值升高,差异有显著性意义(P<0.05)。实验结果表明,对于新型超声处理活检标本,应优先选择存储<1年的样本进行日常分子检测,储存3年内的样本可满足二代测序等检测要求,5年内样本仅可尝试进行PCR等检测,存储超过5年的样本不建议进行后续分子检测。

板栗叶片DNA的提取及AFLP反应体系的建立

以初展开的板栗嫩叶为材料,利用改进的CTAB法,提取到高质量的板栗叶片总DNA。通过优化酶切连接、预扩增、选择性扩增等试验条件,建立了板栗AFLP银染反应体系,得到了清晰的板栗AFLP指纹图谱。为板栗品种的分子标记和板栗品种间亲缘关系等研究奠定了基础。研究结果表明:①DNA模板的质量影响酶切以及后续的连接扩增反应。以初展开的嫩叶提取的板栗叶片总DNA纯度最高,所含蛋白质、小分子杂质少,改良的CTAB提取法可用于板栗AFLP分析,形成清晰的AFLP指纹。②先进行酶切后再进行酶连的反应体系比酶切酶连一起进行的体系效果更好。板栗基因组DNA最佳酶切反应体系为:模板DNA总量500ng,反应体积为25μl,10×Y+/TanqoTMBuffer2.5μl,BSA0.8μl,EcoRI5U,MseI5U。连接反应体系中T4DNA连接酶浓度2U即达最佳效果。酶切、酶连反应最佳温度均为37℃。③以板栗为材料进行AFLP标记时,E AAC+M CAA、E AAC+M CAT和E AGT+M CAT三个引物均获得较好的多态性。其中以E AGT+M CAT引物组合的扩增条带信号强度一致性好,条带分布均匀,能够得到稳定、清晰、分辨率较高的指纹谱带,可进行中国板栗的遗传变异分析。

Detection of Gene Dosage in Circulating Free Plasma DNA as Biomarker for Lung Cancer

The increase in the number of gene copies at specific loci is a genetic alteration frequently associated with over expression of the related protein in cancer cells. Genes whose dose is consistently augmented in cancer include those involved in cell cycle control, proliferation, apoptosis, and angiogenesis among others. In this study, gene dose of onc ogenes MYCL1, MYCN, MYC, EGFR, ERBB2 and AKT2 in DNA obtained from lung tissue and blood plasma, of patients with primary lung cancer was evaluated with respect to normal lung tissue and plasma DNA of healthy individ uals, to determine the capacity of these genes to discriminate normal and neoplastic phenotypes. The number of copies of each gene was determined using real-time (2-△△CT). The AKT2 oncogene was found to be amplified frequently in plasma DNA from patients (74% of cases). This marker showed a noticeable ability to discriminate normal and neo-plastic phenotypes, with a 76 to 89% probability of correctly recognize a plasma sample provided by a lung cancer patient or a healthy individual. For this reason, this detection could be a very useful tool to supplement the existing diagnostic methods in pulmonary cancer.

基于活产建立体外受精-胚胎移植精子DNA碎片指数的参考阈值及子代短期安全性

背景:精子DNA碎片指数与受精、胚胎发育潜能、胚胎植入、流产及子代安全性等存在显著的相关性。然而,其临床参考值受多种因素的影响,导致临床意义极其有限,该研究以活产为结局,通过倾向评分匹配校正其他混杂因素后,构建精子DNA碎片指数与活产的最佳临床截断值,并对其进行内外部验证,具有较好的预测价值及临床应用效能。目的:探讨基于活产建立体外受精-胚胎移植精子DNA碎片指数的参考阈值及子代短期安全性。方法:选取2019年5月至2021年5月于常州市妇幼保健院接受体外受精-胚胎移植患者1921例,以倾向匹配容差0.02为标准,1∶1进行倾向评分匹配,结果活产组与非活产组各成功匹配540例,以此建立模型组;通过选取同时期广西壮族自治区南溪山医院接受体外受精-胚胎移植患者135例作为外部验证组;采用受试者工作曲线探求精子DNA碎片指数对活产的临床最佳截断值,分别采用限制性立方样条曲线、标准曲线、临床决策曲线、临床影响曲线及内外部验证等方法,对该截断值的准确性及临床应用效能进行评估。结果与结论:(1)非活产组精子DNA碎片指数显著高于活产组且与活产存在显著的负相关性(r=-0.444,P<0.001);(2)受试者工作曲线结果显示,DNA碎片指数对活产的最佳截断值为24.33%,曲线下面积为0.775(0.746,0.804),特异度为72.60%,敏感度为78.90%,准确度为75.70%;(3)限制性立方样条曲线拟合Logistic回归结果显示,当精子DNA碎片指数大于24.57%时,临床非活产的风险呈趋势性增涨;(4)Logistic回归概率分析结果显示,精子DNA碎片指数为活产的危险因素[OR(95%CI)=0.916(0.904,0.928),P<0.001],且当精子DNA碎片指数大于27.78%时,临床活产发生的概率将小于50%,随着精子DNA碎片指数每增高1个单位,活产的概率下降8.4%;(5)内外部对该临床截断值的验证均显示,该截点具有一定的临床预测价值及准确性;(6)临床决策曲线与临床影响曲线显示,以该临床截断值建立的预测模型在阈概率为0.22-0.73时具有临床最大净获益值,且在该阈概率范围内损失与获益的比值始终小于1,证实该预测模型具有较好的临床应用效能;(7)精子DNA碎片指数与子代短期安全性分析结果显示,精子DNA碎片指数与出生儿早产、体质量、畸形、性别差异无显著性;(8)结果表明,精子DNA碎片指数对体外受精-胚胎移植活产的最佳临床截断值为24.33%,以此建立的临床预测模型具有较好的区分度、准确度与临床应用效能,精子DNA碎片指数对子代短期安全性影响并不显著,但仍需大样本及长期的追踪评估。

FTA-环介导等温扩增技术直接提取变异链球菌DNA的效果评价

背景:变异链球菌是龋病的重要病原菌,及时检测变异链球菌水平对龋病的早发现、早治疗有重要意义。目的:建立并评价FTA-环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术直接提取变异链球菌DNA的应用效果。方法:①制备含有ATCC标准菌株变异链球菌的菌悬液,接种于脑心浸出液培养基,充分混匀后按10倍梯度稀释成7种浓度(4.2×10^(7),4.2×10^(6),4.2×10^(5),4.2×10^(4),4.2×10^(3),4.2×10^(2),4.2×10 CFU/mL),每个稀释级做2个平行对照,并增加无菌水作为空白对照;②分别采用FTA卡、常规煮沸法、试剂盒提取及裂解液提取4种方法直接提取菌株DNA,通过LAMP技术进行扩增,并进行特异性试验,比较4种提取方法的差异。结果与结论:①4种方法提取的DNA均满足LAMP扩增的要求;②特异性试验结果显示,只有变异链球菌才可特异扩增出靶基因;③裂解液提取法最低检测限为4.2×10^(3) CFU/mL,FTA卡提取法最低检测限为4.2×10^(4) CFU/mL,试剂盒提取法和常规煮沸法最低检测限分别为4.2×10^(6) CFU/mL和4.2×10^(7) CFU/mL;④4种提取方法其他方面的比较显示,试剂盒提取法的实验成本、步骤数和时间都是最高;其他3种方法步骤数一致,其中FTA卡所需仪器设备最少,常规煮沸法单次成本最低,裂解液提取法所需时间最少;FTA卡和裂解液提取法仅需少量菌即可提取成功,后者在时间方面优于FTA卡,但相较于FTA卡其单次成本高,所需设备多;⑤结果说明,该研究建立的FTA-LAMP技术具有操作简便、特异性强、灵敏度高、结果可视化等优势,有望为高效提取检测变异链球菌提供新途径。

DNA存储技术:挑战与未来

随着全球数据呈现指数级增长,当前的信息存储技术面临维护成本高昂、存储寿命有限等多个缺陷,逐渐无法满足日益凸显的需求。因此,迫切需要引入新的信息存储方法来解决这一问题。DNA作为一种天然的遗传信息载体,具备高存储密度、潜在低维护成本和长寿命等优势,因此被视为一种有潜力的新型信息存储介质。该文对DNA数据存储技术的基本原理和流程进行了概述,并回顾了其历史发展。同时,对当前基于DNA存储的领域仍面临的挑战进行了总结,如缓慢的数据写入和读取速度等,以及应对这些挑战的一些潜在策略。最后,为了满足全球对新存储方法的需求,该文指出了DNA数据存储技术的未来发展方向。

DNA存储系统中的数据写入

世界的数字化给人们的生活带来了极大的变化,但与此同时,史无前例的数据激增使得信息存储面临的挑战日益严峻。随着全球数据总量的指数级增长,传统存储介质将无法满足数字化带来的存储需求。使用DNA分子作为基本载体的信息存储展现出高存储密度、低维护成本和易于化学修饰等独特优势。DNA存储主要包括编码、写入、保存、检索、读取和解码六个主要步骤,其中数据的写入是实现DNA存储功能的基础。本文首先介绍DNA存储系统中体外写入数据的策略方法,主要分为将数据写入DNA序列和写入DNA结构两个部分,接着概述体内写入数据技术的发展,最后将讨论DNA存储系统中数据写入面临的写入成本高、写入速度慢等挑战,并对大规模合成高纯度DNA、改进生物酶等具有前景的应用技术进行展望。