Reliability of gender determination using the polymerase chain reaction (PCR) for single cells

Contamination with extraneous DNA sequences is a frequent problem when performing PCR analysis of single cells. This report describes our experience with eliminating contaminating DNA sequences from PCR reagents for the purposes of gender identification. We have used amplification of Y-specific sequences to identify the gender of single human amniocytes. Female cells consistently showed no Y-specific bands but only 80% of male cells showed the expected intense Y-specific band. This phenomenon could lead to incorrect gender identification of single cells. We developed a technique of simultaneous amplification of X- and Y-specific sequences to prevent misdiagnosis because of failed PCR, which allows accurate preimplantation gender determination for women at risk for conceiving children with X-linked genetic discoses. We analyzed the gender of 141 consecutive single cells in a blinded manner without a single incorrect gender assignment     

Identifying the sex of human preimplantation embryos in x-linked disease: Amplification efficiency of a Y-specific alphoid repeat from single blastomeres with two lysis protocols

Introduction: Preimplantation diagnosis involves detecting genetic defects in one or two blastomeres biopsied from cleavage stage embryos followingin vitro fertilization (IVF). For X-linked recessive disease, identification of the sex of embryos allows transfer of only unaffected females. To examine how critical the preparation of the single blastomere is for amplification of a Y chromosome specific repeat sequence using the polymerase chain reaction (PCR), the incidence of amplification failure has been examined following two lysis protocols. Materials and Methods: Amplification of a Y alphoid repeat sequence from single blastomeres disaggregated from cleavage stage embryos was examined after either (1) lysis in distilled water and freeze-thawing twice or (2) a two-step lysis protocol involving an initial treatment in potassium hydroxide and dithiothreitol. Some of the embryos had been previously sexed by cleavage-stage biopsy and fluorescent in situ hybridization with X- and Y-specific probes. Results: Amplification failure occurred in 6 of 50 (12%) and 4 of 60 (7%) single blastomeres from male embryos following lysis in distilled water or using the two-step protocol, respectively. Conversely, amplification from contaminating DNA occurred in 5 of 63 (8%) single blastomeres from female embryos and 6 of 94 (6%) of control medium blanks. Conclusions: The incidence of amplification failure was improved but not eliminated using the two-step lysis protocol. At least two cells, therefore, would be necessary for accurate identification of males by amplification of Y-specific repeat sequences alone. Nevertheless, this protocol for preparing cleavage-stage blastomeres is likely to give more consistent amplification of any unique or repeat sequences.Presented at the 5th Annual Meeting of the International Working Group on Preimplantation Genetics, Hamburg, Germany, June 28, 1995.     

Detection of human papillomavirus DNA in cervical smears by the polymerase chain reaction

Abstract. Chetsanga C, Pettersson B, Pettersson U, Gyllensten U. Detection of human papillomavirus DNA in cervical smears by the polymerase chain reaction. Int J Gynecol Cancer 1991; 1 : 209–213.
A PCR-based method has been developed which allows human papillomavirus DNA to be detected in routinely obtained cervical smear specimens. The results showed that 76% of the smears that were collected from women who were carrying HPV-positive lesions by cervical swab analysis were positive by the PCR-based method. The results suggest that it is possible to carry out population-based retrospective studies of archival cervical smears.     

Reliability of polymerase chain reaction (PCR) analysis of single cells for preimplantation genetic diagnosis

Purpose We investigated the reliability of polymerase chain reaction (PCR) genotype analyses performed on single cells for the purposes of preimplantation genetic analysis.Methods We performed blind analysis of 130 single skin fibroblasts heterozygous for the -F508 mutation in the cystic fibrosis transmembrane regulator (CFTR) gene and 73 single skin fibroblasts from an individual heterozygous for the Xbapolymoporphic site of the Factor VIII gene.Results Amplification was successful for 116 cells and 52 cells respectively and in all but one case (a CFTR analysis) both alleles were amplified. The incidence of diagnostic error was 1 out of 203 analyses or 0.0043. We conclude that PCR is a reliable method for determining the genotype of single cells for the purposes of preimplantation genetic analysis.     

Uptake of exogenous human papilloma virus L1 DNA by oocytes and detection by the polymerase chain reaction

Objective The purpose of this study was to determine if oocytes were capable of taking up exogenous DNA such as human papillomaviral (HPV) DNA and evaluate the zona pellucida as a barrier to the entry of foreign DNA into the oocyte.Methods The experiment consisted of four groups of hamster oocytes exposed to HPV DNA fragments: Group A, zona-free oocytes (n =5); Group B, oocytes with an intact zona pellucida (n =5); Group C, oocytes fixed in 4% buffered formalin solution for 20 min (n =5); and Group D, zona-free oocytes (n =4). Group C oocytes served as an internal control to ensure adequate washing of the oocytes after incubation. Results The zona pellucida was not a barrier to foreign DNA molecules. The PCR did not detect L1-HPV and -globin gene sequences in the untreated hamster oocyte. Uptake of the smaller DNA fragments such as that amplified from the -globin region was independent of active oocyte cell processes. Conclusion Oocytes cultured in vitro can passively take up exogenous DNA fragments. The results suggest a possible role of oocytes as vectors for foreign DNA.     

Influence of mosaicism on sexing of human preembryos detected by the polymerase chain reaction

Purpose: Preimplantation sex determination using a single cell by the polymerase chain reaction (PCR) was investigated to elucidate the influence of mosaicism. Methods: The SRY and ZFX genes were coamplified as target sequences for the Y and X chromosomes, respectively. The sensitivity of the single and nested PCR method was examined initially followed by amplification of single amniocytes by the nested PCR. Then the sex of single blastomeres at the three- and nine-cell stages was determined by the nested PCR. Results: The nested PCR was 10⁴-fold more sensitive than the single PCR. Sex determination was possible in 97.5% (117/120) of the blastomeres tested. However, the correspondence rate for all blastomeres within a single embryo was only 60% (12/20 embryos). Among the remaining embryos for which sexing of all blastomeres was not consistent, only one blastomere showed findings indicating the presence of mosaicism (or pseudomosaicism). Conclusions: At least two blastomeres need to be assessed when determining the sex of an embryo in order to avoid misdiagnosis due to mosaicism (or pseudomosaicism).     

应用PCR技术对输卵管妊娠患者性传播疾病病原体感染的研究

目的探讨输卵管妊娠(TP)患者性传播疾病(STD)病原体的感染情况。方法2001年1月至2002年12月间,东莞市人民医院及中山大学东华医院,应用聚合酶链反应技术对249例TP患者的宫颈分泌物及子宫内膜活检标本进行了7种常见STD病原体的检测。结果解脲支原体(UU)的检出率最高,达27.71%和23.29%,其次为沙眼衣原体(CT)20.88%和17.27%,与对照组比较差异有显著性(P<0.05),淋球菌(NG)检出率为7.63%和8.84%、乙型肝炎病毒(HBV)检出率6.02%和4.81%、乳头状瘤病毒(HPV)检出率为3.61%和4.02%、单纯疱疹病毒(HSV)检出率为2.41%和1.20%、梅毒螺旋体(TMP)检出率为0.40%和0。同时还发现各种病原体的上行性感染及混合感染情况。结论与TP相关的病原体中以UU最多见,各种病原体混合感染占一定比例。     

Rapid detection of trisomy 21 by gene dosage analysis using quantitative real-time polymerase chain reaction

AIM: Rapid detection of fetal aneuploidy helps inform a mother's choice about the course of her pregnancy. Obtaining results by fluorescent in situ hybridization (FISH) requires more than 24 h, and thus a more rapid method is needed. METHODS: Conventional G-banding and FISH for chromosome 21 were performed for cultured amniocytes. Genomic DNA was extracted from uncultured amniocytes obtained from 23 patients. TaqMan polymerase chain reaction (PCR) primers were designed to amplify the potassium voltage gated channel gene on chromosome 21q22.12 and the ribosomal phosphoprotein gene on 18q21.1. Quantitative real-time PCR was performed for these two gene fragments and the differences of the threshold cycle (Ct) of the two genes (Ct 18-Ct 21) were calculated for each sample. RESULTS: G-banding revealed that 19 patients had a normal karyotype and four had trisomy 21. FISH resulted in one case of a false positive. The Delta Ct values (Ct 18-Ct 21) of trisomy 21 patients were significantly higher than the values of individuals with normal karyotypes (P < 0.001) and there was no overlapping. CONCLUSIONS: Fetal trisomy 21 is rapidly detectable by gene dosage analysis from amniocytes using quantitative real-time PCR.     

Quantitative fluorescent polymerase chain reaction to detect chromosomal anomalies in spontaneous abortion

OBJECTIVES: To evaluate the value of short tandem repeats (microsatellites) in the study of numerical chromosomal anomalies in spontaneous abortion. METHOD: Multiplex quantitative fluorescent polymerase chain reaction (QF-PCR) was carried out on 61 spontaneous abortion samples and 48 controls using microsatellite markers from 8 chromosomes where aneuploids are commonly found. RESULTS: Of the 61 samples, 65.6% were successfully karyotyped, and the call rate of the QF-PCR was 98.3%. The correspondence between PCR and karyotyping was 95%. The success rate of karyotyping in the inevitable abortion group was 79.6%, higher than for the missed abortion group (8.3%), P<0.001. The call rate of QF-PCR showed no difference between these 2 groups (100% vs 91.7%, P=0.197). CONCLUSION: Microsatellite-based QF-PCR is a helpful and reliable tool to diagnose numerical chromosomal anomalies in spontaneous abortion. It also provides a diagnosis for necrotic tissue.     

Intrapartum group B Streptococcus screening using real-time polymerase chain reaction in Japanese population

Objective: The objective of this study was to analyze the diagnostic accuracy of a commercial real-time polymerase chain reaction (PCR) assay for group B streptococcus (GBS) colonization status and to compare results of the intrapartum PCR with the antepartum conventional GBS culture in Japanese pregnant women.

Methods: This prospective observational study enrolled Japanese pregnant women at 35–37 weeks’ gestation. Paired recto-vaginal swabs were obtained for PCR and conventional culture, both at 35–37 weeks’ gestation and at admission for delivery. Performance of PCR was analyzed by comparing with the culture results. Furthermore, using the intrapartum culture results as the gold standard, the test of both the antepartum culture and the intrapartum PCR were characterized.

Results: We prospectively enrolled 79 pregnant women at 35–37 weeks’ gestation, and the intrapartum results were obtained from 73 of those women. The sensitivity of PCR was 86.2%, and concordance rate with the conventional culture was 96.7% overall. Compared with the intrapartum culture, the sensitivity and the specificity of the intrapartum PCR were 83.3% and 98.4%, respectively, while the sensitivity and the specificity of the antepartum culture were 100.0% and 95.1%.

Conclusions: The intrapartum real-time PCR assay for GBS screening has the accuracy similar to the antepartum conventional culture method.     

Cryopreservation of blastomeres separated from two-cell mouse embryos by an ultrarapid freezing method

Purpose To clarify the developmental capacity of frozen two-cell blastomeres, we investigated in vivo and in vitro viabilities of blastomeres that were frozen ultrarapidly after separation from two-cell mouse embryos. Two-cell embryos obtained from superovulated F₁ hybrid females were denuded by treatment with 0.5% pronase solution and then induced to separate into two single blastomeres by gentle pipetting. The blastomeres were cryopreserved by an ultrarapid freezing method.Results The preimplantation developmental rate of two-cell embryos frozen in 3.0 MDMSO was significantly higher than the rate of those frozen in 15 and 4.5 MDMSO (at least P<0.05). The in vitro developmental rate of the ultrarapidly frozen-thawed blastomeres separated from two-cell embryos (75.0%) was similar to that of nonfrozen blastomeres (76.0%). When eight pairs of blastocysts that developed from frozen two-cell mouse blastomeres were transferred to pregnant ICR recipients on Day 3, four live singletons were born.Conclusion Thus, the results indicate that two-cell mouse blastomeres can be frozen by the ultrarapid freezing method.     

Usefulness of quantitative polymerase chain reaction in amniotic fluid as early prognostic marker of fetal infection with Toxoplasma gondii

OBJECTIVE: Our purpose was to evaluate Toxoplasma gondii concentration in amniotic fluid (AF) samples as a prognostic marker of congenital toxoplasmosis. STUDY DESIGN: A retrospective study was carried out in 88 consecutive AF samples from 86 pregnant women, which were found positive by prospective polymerase chain reaction (PCR) testing. Parasite AF concentrations were estimated by real-time quantitative PCR and analyzed in relation to the clinical outcome of infected fetuses during pregnancy and at birth, taking into account the gestational age at maternal infection. RESULTS: A significant negative linear regression was observed between gestational age at maternal infection and T gondii DNA loads in AF. After adjusting for time at maternal seroconversion by multivariate analysis, higher parasite concentrations were significantly associated with a severe outcome of congenital infection (odds ratio [OR]=15.38/log (parasites/mL AF) [95% CI=2.45-97.7]). CONCLUSION: PCR quantification of T gondii in AF can be highly contributive for early prognosis of congenital toxoplasmosis. Maternal infections acquired before 20 weeks with a parasite load greater than 100/mL of AF have the highest risk of severe fetal outcome.     

荧光定量PCR检测孕妇血浆中胎儿DNA的研究

目的探讨荧光定量PCR（fluorescence quantitative PCR，FQ-PCR）方法检测孕妇血浆中胎儿DNA的可行性，探讨胎儿DNA在孕妇血浆中的含量及其在孕期的变化规律。方法以胎儿SRY基因序列作为胎儿DNA在孕妇血浆中的标志，应用荧光MGB（Minor Groove Binder）探针实时定量PCR方法连续测定30例孕妇在不同孕期和产后共237份血浆标本中胎儿DNA的含量。结果使用该方法最低可检测到20000个女性DNA中的一个男性DNA。孕妇血浆中最早检出SRY序列的时间为孕6^＋6周。孕8周起，所有怀男胎孕妇的标本中均可检出SRY序列，其含量随着妊娠的进展而增高，在晚期妊娠达高峰，产后24～48h血浆中SRY序列检测均为阴性。胎儿DNA在孕妇血浆总DNA含量中的相对浓度分别为孕早期4．88％、孕中期6．10％、孕晚期4．77％。全部实验无假阳性和假阴性出现。结论FQ-PCR方法是一种灵敏度和准确性高，特异性强的定量检测孕妇血浆中胎儿游离DNA的方法，孕妇血浆中存在高浓度的胎儿DNA，可用于无创伤性产前基因诊断。     

Accuracy of a rapid real-time polymerase chain reaction assay for diagnosis of group B Streptococcus colonization in a cohort of HIV-infected pregnant women

Objective: There are limited data regarding Xpert performance to detect Group B Streptococcus (GBS) in HIV-infected pregnant women. We evaluated the accuracy of a rapid real-time polymerase chain reaction (PCR) test in a cohort of HIV-infected women.

Methods: At 35–37 weeks of pregnancy, a pair of combined rectovaginal swabs were collected for two GBS assays in a cohort of sequentially included HIV-infected women in Rio de Janeiro: (1) culture; and (2) real-time PCR assay [GeneXpert GBS (Cepheid, Sunnyvale, CA)]. Using culture as the reference, sensitivity, specificity, positive and negative-likelihood ratios were estimated.

Results: From June 2012 to February 2015, 337 pregnant women met inclusion criteria. One woman was later excluded, due to failure to obtain a result in the index test; 336 were included in the analyses. The GBS colonization rate was 19.04%. Sensitivity and specificity of the GeneXpert GBS assay were 85.94% (95% CI: 75.38–92.42) and 94.85% (95% CI: 91.55–96.91), respectively. Positive and negative predictive values were 79.71% (95% CI: 68.78–87.51) and 96.63% (95% CI: 93.72–98.22), respectively.

Conclusions: GeneXpert GBS is an acceptable test for the identification of GBS colonization in HIV-infected pregnant women and represents a reasonable option to detect GBS colonization in settings where culture is not feasible.     

应用套式聚合酶链反应技术检测孕妇与胎儿巨细胞病毒感染

目的评价套式聚合酶链反应技术（ＮＴ－ＰＣＲ）加限制酶分析在孕妇与胎儿人巨细胞病毒（ＨＣＭＶ）感染检测中的应用价值。方法应用ＮＴ－ＰＣＲ及限制酶分析、病毒分离、电镜观察和特异性抗体测定,对各孕期孕妇３６７例的外周血、部分孕妇脐血及死胎组织进行ＨＣＭＶ检测。结果孕早、中、晚期ＨＣＭＶ阳性检出率分别为８．６％、１．６％及７．０％,ＮＴ－ＰＣＲ检出率（４．９％）高于病毒分离（３．０％,Ｐ＜０．０２５）。６份ＨＣＭＶＤＮＡ阳性母血中,３份配对脐血ＨＣＭＶＤＮＡ也阳性。其中２对ＮＴ－ＰＣＲ、病毒分离及特异性ＩｇＭ、ＩｇＡ均阳性,１对ＮＴ－ＰＣＲ、病毒分离、特异性ＩｇＡ阳性,而ＩｇＭ阴性。２８例死胎组织中,发现１例死胎肺组织ＨＣＭＶＤＮＡ阳性。结论ＮＴ－ＰＣＲ能提高诊断ＨＣＭＶ感染的特异性与敏感性     

用多聚酶链反应探讨单纯疱疹病毒感染与宫颈疾病的关系

使用多聚酶链反应（ＰＣＲ）探讨了单纯疱疹病毒感染与慢性宫颈炎，宫颈癌的关系。结果表明，在３１份正常妇女，７２份慢性宫颈炎、２１份宫颈癌标本中检出ＨＳＶ阳性分别为４份（１３．３％）、２５份（３４．７％）和１２份（５７．１％），阳性率呈明显升高的趋势；在慢性宫颈炎患者中，随宫颈糜烂程度的增加，ＨＳＶ感染率升高。提示ＨＳＶ感染与慢性宫颈炎、宫颈癌的发生、发展相关；ＰＣＲ是检测ＨＳＶ感染灵敏、简便的方法。     

尖锐湿疣和假性湿疣病理组织学及人乳头瘤病毒DNA研究

应用免疫组化、核酸原位杂交和聚合酶链的（ＰＣＲ）等方法，对２７４例尖锐湿疣和假性湿疣病例的活检组织石蜡包埋标本，进行病理组织学和人乳头瘤病毒（ＨＰＶ）６ｂ，１１ＤＮＡ及核壳抗原（ＨＰＶ－Ａｇ）的检测，结果：尖锐湿疣和假性湿疣表面上皮呈乳头状结构，尖锐湿疣有明显的基底细胞增生，上皮增厚，典型的挖空细胞，假性湿疣则无上述改变。尖锐湿疣组织中ＨＰＶ６ｂ，１１ＤＮＡ检测阳性率分别为：核酸原位杂交法８２．５