Detection of porcine reproductive and respiratory syndrome virus in boar semen by PCR.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes a devastating disease in swine. The presence and transmission of PRRSV by boar semen has been demonstrated by using a swine bioassay. In this assay, 4- to 8-week-old pigs were inoculated intraperitoneally with semen from PRRSV-infected boars. Seroconversion of these piglets indicated the presence of PRRSV in semen. Seroconversion in gilts has also been demonstrated following artificial insemination with semen from PRRSV-infected boars. These methods of detecting PRRSV in boar semen are time-consuming, laborious, and expensive. The objective of this study was to develop a reliable and sensitive PCR assay to directly detect PRRSV in boar semen. Primers from open reading frames 1b and 7 of the PRRSV genome were used in nested PCRs. Virus was detected at concentrations as low as 10 infectious virions per ml in PRRSV-spiked semen. Specificity was confirmed by using a nested PCR and a 32P-labeled oligonucleotide probe. The primers did not react with related arteriviruses or other swine viruses. The PCR assay showed good correlation with the swine bioassay, and both methods were superior to virus isolation. To consistently identify PRRSV in boar semen, the cell fraction was separated by centrifugation at 600 x g for 20 min, a lysis buffer without a reducing agent (2-mercaptoethanol) was used, and nondiluted and 1:20-diluted cell fractions were evaluated by PCR. PRRSV was not reliably detected in the seminal plasma fraction of boar semen.     

人类精液一步冷冻法的研究

本文应用聚丙烯1ml注射器作冻贮容器实验人类精液的一步冷冻法。结果表明,正常生育力组(n=45)和不育症组(n=50)的精子冷冻复苏率分别为69.4±9.7%、58.0±14.7%;一步冷冻组与程控仪冷冻组(n=14)的冷冻精子活率比较、与未经冷冻组(n=10)的去透明带地鼠卵穿透试验比较没有显著性差异(P>0.05);冻后精子体外能存活较长时间;37℃水浴解冻有利于精子存活。提示一步冷冻人类精液可获得良好的冷冻效果。     

PCR detection and evidence of shedding of porcine circovirus type 2 in boar semen

An experimental study was conducted to evaluate the potential presence of porcine circovirus type 2 (PCV2) in the semen of infected boars. Four mature boars were inoculated intranasally with PCV2 isolate LHVA-V53 propagated on PK15 cells. Two boars inoculated with the supernatant of noninfected PK15 cells were kept as controls. Serum samples were collected from all boars at 4, 7, 11, 13, 18, 21, 25, 28, 35, and 55 days postinoculation (dpi) and from the four PCV2-infected boars at 90 dpi. Samples were tested for the presence of antibodies to PCV2 by an indirect immunofluorescence assay and for the presence of PCV2 DNA by PCR and nested PCR. Semen samples were collected from all six boars at 5, 8, 11, 13, 18, 21, 25, 28, 33, and 47 dpi and tested for the presence of PCV2 DNA by a nested PCR assay. Antibodies to PCV2 could be detected as early as 11 dpi in one boar, and all four infected boars were found positive for PCV2 antibodies by 18 dpi. Thereafter all infected boars remained positive for antibodies to PCV2 until 90 dpi. Analysis of serum samples by nested PCR demonstrated the presence of PCV2 DNA as early as 4 dpi in three of four infected boars. Serum samples from all infected boars were positive for PCV2 DNA from 11 dpi until 35 dpi but were negative at 90 dpi. PCV2 DNA was detected as soon as 5 dpi in the semen of two infected boars and intermittently thereafter in the semen of all four infected boars. The semen of two infected boars was positive for PCV2 DNA at 47 dpi. Following infection, PCV2 DNA can be detected in semen concurrently with the presence of PCV2 DNA and antibodies in the serum. The present study suggests that PCV2 may be shed intermittently in the semen of infected boars.     

精浆抗精子抗体对男性不育症患者精液参数的影响分析

目的分析男性不育症患者精浆抗精子抗体对精液相关参数的影响.方法收集男性不育症患者260例,均采用免疫珠法测定精浆抗精子抗体(AsAb,IgA或IgG型),根据结果分为AsAb阳性组(41例)和阴性组(219例).结果在这260例男性不育症患者中精浆抗精子抗体检出率为15.77%.AsAb阳性组患者的精液主要参数(精液密度、活动率、a b活动力)均低于AsAb阴性组患者,两组间比较差异具有显著性(P<0.05).结论精浆抗精子抗体对精液主要参数有明显影响,是导致男性免疫性不育的重要因素之一.     

Detection of hemizygosity in Hermansky-Pudlak syndrome by quantitative real-time PCR

Hermansky-Pudlak syndrome (HPS) is an autosomal recessive disorder characterized by oculocutaneous albinism, a bleeding diathesis and, in some patients, pulmonary fibrosis or granulomatous colitis. HPS is associated with biosynthesis defects of melanosomes, platelet-dense bodies, and lysosomes. There are seven genetic HPS subtypes; HPS-1 is the most common. We used a real-time quantitative PCR (qPCR) approach to investigate six HPS-1 patients, previously assigned as having homozygous mutations in the HPS1 gene. HPS1 gene copy numbers, calculated by use of a comparative Ct method, revealed that one patient was in fact hemizygous for her c.1189delC (S396delC) HPS1 mutation. The causative deletion/insertion was 13,966 bp in size, with defined breakpoints, and involved an adjacent gene (C10orf33). A mechanism of formation is proposed for the deletion/insertion, and both multiplex and qPCR indicated that the deletion/insertion was present in the patient, her brother, and her father. qPCR amplification is valuable for detecting deletions too small to be identified by fluorescence in situ hybridization. This demonstration of hemizygosity, performed using genomic DNA, can eliminate concerns about non-paternity and can verify the diagnosis of an autosomal recessive disorder when a DNA alteration appears to be homozygous by standard PCR and sequencing methods, and its pathogenicity is in doubt.     

荧光定量聚合酶链反应检测乙型肝炎病毒DNA

目的 建立检测HBV病毒DNA的荧光定量PCR法（FQ-PCR）,并与运用常规凝胶电泳技术观察特异扩增带检测的结果加以比较。方法 合成扩增HBV DNA314bp特异保守序列的1对引物及1条带2个荧光基团的寡核苷酸探针。用PE-5700型定量PCR仪完成PCR反应及产物的荧光定量检测。同是PCR产物经琼脂糖凝胶电泳,EB染色,UVP（凝胶成像仪）检出有314bp带者为阳性或弱阳性。结果 建立了检测HBVDNA的荧光定量PCR技术,用已知HBV阳性模板不同拷贝数的标准溶液测得标准曲线Ct,原始拷贝数在10^5/ml以上者为阳性,用定量及定性PCR2种方法检测698例血清标本的结果表明：用FQ-PCR技术共检测出204例为阳性,阳性率29.2%;用定量及定性PCR2种方法检测698例血清标本的结果表明：用FQ-PCR技术共检测出204例为阳性,阳性率29.2%;用定性PCR观察到193例有阳性特异带,阳性检出率为27.65%。没有发现用定性PCR检测为阳性而用FQ-PCR检测为阴性者。结论 FQ-PCR检测HBVDNA较普通定性PCR技术具有操作简便,灵敏度更高、减少发生污染可导致假阳性结果的可能性及自动化程度高等优点,值得在临床检验中推广应用。     

Detection of Gallibacterium anatis by TaqMan fluorescent quantitative PCR

To better understand the prevalence of Gallibacterium anatis in different poultry species, a rapid and accurate method was developed to detect G. anatis using a TaqMan fluorescent quantitative polymerase chain reaction (qPCR). Specific primers and a TaqMan probe were designed based on the reference gtxA gene sequence. The qPCR standard curve showed a good linear relationship, and the method showed good reproducibility, sensitivity, and specificity, indicating its suitability for G. anatis identification and quantitative analysis. A comparison of the detection results in 160 clinical swab samples showed that the detection rate (54.4%) of the qPCR for G. anatis was better than that of two conventional methods: gyrB gene-based qPCR for G. anatis (51.9%) and culture-based identification (34.4%). G. anatis was detected in layer chicken (77.3%), Silkie chicken (72.7%), and duck (27.1%) with relatively high detection rates, whereas dove (8.8%) and quail (3.0%) showed lower detection rates, indicating the different prevalence of G. anatis in different fowl species.     

Detection of human cytomegalovirus DNA by real-time quantitative PCR

A real-time PCR assay was developed to quantify human cytomegalovirus (CMV) DNA. This assay was used to demonstrate a higher CMV DNA load in plasma of bone marrow transplant patients than in that of blood donors. The CMV load was higher in CMV antigen-positive patients than in antigen-negative patients.     

应用SELDI技术检测精索静脉曲张患者精浆中的差异表达蛋白

目的探讨用蛋白质芯片技术筛选精索静脉曲张患者精浆中蛋白质表达谱,寻找差异蛋白。方法采用表面增强激光解离飞行时间质谱技术（surface—enhanced laser desorption／ionization time of flight mass spectrometry,SELDI—TOF—MS）．运用CM10蛋白质芯片检测30例精索静脉曲张患者和30例正常对照精浆中蛋白质谱,获得的蛋白质谱采用Biomarker Wizard软件分析,初步筛选蛋白质峰,结合生物信息学的支持向量机（support vector machines,SVM）方法建立并测试精索静脉曲张患者精浆中的蛋白质指纹图谱。结果在芯片上捕获到163种蛋白质,用质谱仪筛选出精索静脉曲张患者与正常对照组相比的16种差异蛋白,其中有3个蛋存在显著性差异。结论精索静脉曲张患者与健康者精浆中存在较多差异蛋白质,本研究对进一步探索精索静脉曲张的病因及其临床治疗具有重要意义。     

多重PCR检测儿童呼吸道感染常见病毒

目的 对深圳、粤东地区儿童呼吸道病毒感染进行监测、分析,探讨其流行规律.方法 2006年6月-2008年6月,于汕头大学附属医院、深圳市第四人民医院、揭阳市人民医院采集毛细支气管炎、支气管肺炎、喘息支气管炎等的患儿咽拭子或鼻咽分泌物标本686份,多重PCR进行9种呼吸道病毒检测.结果 在686例标本中病毒阳性362例(52.77%),其中呼吸道感染患儿中呼吸道合胞病毒(RSV)感染占首位,达到31.22%(113/362),其次是鼻病毒(RHV)为16.85%(61/362),最低是流感病毒B型(IVB)占0.83%(3/362),流感病毒A型(IVA)占14.36%(52/362),副流感病毒1型(PIV1)和副流感病毒3型(PIV3)分别占7.73%(28/362)和8.29%(30/362),腺病毒(AdV)达到9.67%(35/362),而人博卡病毒(hBOV)和人偏肺病毒(hMPV)分别占6.08%(22/362)和4.97%(18/362).结论 RSV、RHV及IVA是华南地区儿童呼吸道感染的主要病毒病原,混合感染以RSV、IVA联合其他病毒感染为主,诊治时应根据所感染病原体制定有针对性的措施.     

Detection of beta-herpesviruses in allogenic stem cell recipients by quantitative real-time PCR

The aim of this study was to investigate the clinical impact of reactivation of human herpes virus-6 (HHV-6) and HHV-7 infections in stem cell transplantation recipients, and to examine a possible increase in virulence of the two roseoloviruses when a reactivation of CMV (HHV-5) simultaneously occurs. For this purpose, quantitative real-time PCR systems were developed to assess the viral load of CMV, HHV-6, or HHV-7 in the plasma of haematopoetic stem cell recipients. One hundred and ninety-eight plasma samples from 37 patients who underwent allogeneic stem cell transplantation were tested for CMV, HHV-6, and HHV-7 by a 5'-exonuclease (TaqMan) quantitative real-time PCR. The CMV load obtained by the real-time PCR assay was compared retrospectively with results generated previously with a commercially available test (COBAS AMPLICOR CMV MONITOR Test, Roche). The results suggest that CMV and HHV-6 may be associated with post-transplantation end-organ disease, while HHV-7 reactivation had no impact on the patients included in this study. No evidence for a potential interaction of the roseoloviruses and CMV infections was found.     

Detection of herpes viruses in clinical samples using real-time PCR

Herpes viruses including cytomegalovirus, varicella zoster and herpes simplex are an important cause of morbidity and mortality, especially in immunocompromised patients. Real-time PCR assays were developed with the aim of introducing a rapid and sensitive test to replace culture, and as a surveillance system for high-risk patients. The assays were optimised using cell culture derived material, and the sensitivity ascertained using cloned product before applying to extracted and non-extracted clinical samples. The sensitivity was between 1--100 virus copies with increased sensitivity to detect less than 10 copies possible when an initial round of amplification was carried out using external primers. Results were available within four hours of receipt compared with a median of 4.4 days for culture and immunofluorescence. Real-time PCR was found to be a sensitive and rapid method of detecting these viruses and will be a valuable tool for the surveillance of immunosuppressed patients.     

运用荧光定量多重PCR技术检测RB1基因突变

目的　建立一种半自动、简易、快速和不需放射性同位素技术,用以检测RB1 基因突变的方法。方法　应用包括扩增RB1 基因27 个外显子和启动区在内的荧光定量多重PCR 技术。将全基因分成若干组,每组3～7 对引物,同时含对照C4 引物并使用4 对外对照,分别为RB的缺体、单体、双倍体及三体。在测试标本中,一个片段的拷贝数根据比较对照与标本的荧光强度而获得。利用自动片段处理软件2.1 处理结果。结果　观察到小片段缺失、插入及外显子拷贝数缺失等突变。测序结果和RB瘤细胞杂合性丢失进一步证实荧光定量多重PCR 的筛查结果。结论　此法是筛查患者基因缺失、插入的一种快速、简易的方法。应用此法可查出约50% 的阳性病例。查出的突变不仅有小缺失、插入,也可发现用以往的方法所不能见到的外显子杂合性缺失病例。对临床基因缺陷的诊断有重要意义     

母体循环中胎儿游离DNA的定量分析

目的依据孕妇血浆中存在游离胎儿DNA的理论，从孕妇外周血浆中分离出胎儿DNA并加以鉴定，预防x连锁遗传病患儿的出生。方法从孕早期、中期共78名孕妇外周血浆中分离胎儿DNA，用实时荧光定量聚合酶链反应（fluorescence quantitative polymerase chain reaction，FQ—PCR）的方法检测其中的Y性别决定区（sex—determining region Y，SRY）基因。结果孕早期怀男胎的孕妇28名，25名SRY基因阳性，其平均浓度为（58．82±25．22）拷贝／ml；孕中期怀男胎的孕妇20名，SRY基因均为阳性，平均为（152．08±62．61）拷贝／ml；怀女胎的孕妇均为阴性。结论用实时荧光定量PCR的方法最早在孕62天的孕妇外周血浆中就可以检测到胎儿SRY基因，随孕周的增加，母血中胎儿DNA的量也在逐渐增加。实时荧光定量PCR技术在进行无创伤性产前性别诊断中有重要的价值。     

用实时荧光定量PCR方法检测母血中的胎儿SRY基因

目的　从孕妇外周血浆中分离出胎儿DNA ,并加以鉴定 ,预防X连锁遗传病患儿的出生。方法　从孕早期、中期共 3 0 0名孕妇外周血浆中分离胎儿DNA ,用实时荧光定量聚合酶链反应 (fluorescencequantitativepolymerasechainreaction ,FQ PCR)的方法检测其中的Y性别决定区 (sex determiningregionY ,SRY)基因。结果　孕早期怀男胎的孕妇 82名 ,70名SRY基因阳性 ,其平均浓度为 ( 5 8.82± 2 0 .90 )拷贝 /ml,中位数为 5 8.5 0拷贝 /ml。孕中期怀男胎的孕妇 90名 ,SRY基因均为阳性 ,平均为 ( 15 2 .0 8± 62 61)拷贝 /ml,中位数为 14 9.3 5拷贝 /ml。怀女胎的孕妇均为阴性。结论　用实时荧光定量PCR的方法最早在孕42天的孕妇外周血浆中就可以检测到胎儿SRY基因 ,随孕周的增加 ,母血中胎儿DNA的量也在逐渐增加。实时荧光定量PCR技术在进行无创伤性产前性别诊断中有重要的价值。     

Rapid typing of influenza viruses using super high-speed quantitative real-time PCR

The development of a rapid and sensitive system for detecting influenza viruses is a high priority for controlling future epidemics and pandemics. Quantitative real-time PCR is often used for detecting various kinds of viruses; however, it requires more than 2h per run. Detection assays were performed with super high-speed RT-PCR (SHRT-PCR) developed according to a newly designed heating system. The new method uses a high-speed reaction (18s/cycle; 40 cycles in less than 20min) for typing influenza viruses. The detection limit of SHRT-PCR was 1 copy/reaction and 10(-1) plaque-forming unit/reaction for viruses in culture supernatants during 20min. Using SHRT-PCR, 86 strains of influenza viruses isolated by the Tokyo Metropolitan Institute of Public Health were tested; the results showed 100% sensitivity and specificity for each influenza A and B virus, and swine-origin influenza virus. Twenty-seven swabs collected from the pharyngeal mucosa of outpatients were also tested, showing positive signs for influenza virus on an immunochromatographic assay; the results between SHRT-PCR and immunochromatography exhibited 100% agreement for both positive and negative results. The rapid reaction time and high sensitivity of SHRT-PCR makes this technique well suited for monitoring epidemics and pre-pandemic influenza outbreaks.     

双重荧光定量RT—PCR在快速检测A、B型流感病毒上的应用

目的 建立双重荧光定量RT-PCR技术同时快速检测A、B型流感病毒,并应用于临床样本的检测.方法 在A型流感病毒M基因和B型流感病毒HA基因的保守区序列分别设计特异性引物和Taqman探针,建立优化双重荧光RT-PCR反应体系,评价所建双重RT-PCR反应体系的特异性、敏感性和稳定性,并应用于疑似流感含漱液标本检测.结果 该方法对A、B型流感病毒检测具有高度特异性,检出限分别为0.1TCID50和0.01TCID50,具有较好的稳定性.可从疑似流感患者含漱液中直接检测到流感病毒核酸.结论 本研究建立的双重荧光定量RT-PCR可以同时准确分型A、B型流感病毒,灵敏度高,稳定性好,是一种快速检测流感病毒的新方法.     

利用精液脱落生殖细胞制备减数分裂中期Ⅰ染色体的研究

本文利用男性不育症精液中的脱落生殖细胞进行了制备减数分裂染色体的研究。在随机连续取样的210例病人中，总共有56例制备出减数分裂染色体；在不同精子密度的病人中，战数分裂染色体检出率分别为：弱精症组33．33%(10／30)，少精症组47．22%(42／88)，无精子症组4．04%(4／99)，减数分裂染色体检出率以伴有多重精子异常的少精症检出率为最高．这类病人可做为精液脱落生殖细胞减数分裂染色体检查的主要适应症，以提高减数分裂染色体的检出率，按本方法制备的染色体83.95%(893／1064)，为质量非常好或可分析的减数分裂染色体。