Reliability of detecting rRNA sequences of Chlamydia trachomatis with fluorescence in situ hybridization without amplification

OBJECTIVE: To use fluorescence in situ hybridization (FISH) using ribosomal RNA (rRNA) oligonucleotide probes as the target nucleic acid for the detection of Chlamydia trachomatis. STUDY DESIGN: Suitable sequences selected from the rRNA sequence of C trachomatis were labeled with a fluorescent dye and used in FISH for detecting chlamydial inclusion bodies and/ or elementary bodies in paraformaldehyde-fixed urogenital swab samples. The sensitivity and specificity of the FISH assay were compared with those of the polymerase chain reaction (PCR) using plasmid primers. Positive known C trachomatis-infected McCoy cells were used as positive controls. Urogenital swab specimens that were C trachomatis negative on culture and PCR were used as negative controls. RESULT: Among the 128 samples included in the study, FISH was positive in 28 (21.8%) and PCR in 33 (25.7%). A significant correlation was found between the 2 detection methods. Results of PCR and FISH were consistent in 115 of the 128 samples (R = 0.89). Thirteen samples showed discordant results. Of these, 9 FISH negative samples were PCR positive and 4 FISH positive samples were PCR negative. CONCLUSION: FISH was a highly specific and fairly sensitive technique for detecting C trachomatis. Signal amplification techniques and use of different fluorophores may further increase the sensitivity of this technique.     

Quantitative PCR in the diagnosis of Leishmania

Polymerase chain reaction (PCR) is a sensitive and rapid method for the diagnosis of canine Leishmania infection and can be performed on a variety of biological samples, including peripheral blood, lymph node, bone marrow and skin. Standard PCR requires electrophoretic analysis of the amplification products and is usually not suitable for quantification of the template DNA (unless competitor-based or other methods are developed), being of reduced usefulness when accurate monitoring of target DNA is required. Quantitative real-time PCR allows the continuous monitoring of the accumulation of PCR products during the amplification reaction. This allows the identification of the cycle of near-logarithmic PCR product generation (threshold cycle) and, by inference, the relative quantification of the template DNA present at the start of the reaction. Since the amplification product are monitored in "real-time" as they form cycle-by-cycle, no post-amplification handling is required. The absolute quantification is performed according either to an internal standard co-amplified with the sample DNA, or to an external standard curve obtained by parallel amplification of serial known concentrations of a reference DNA sequence. From the quantification of the template DNA, an estimation of the relative load of parasites in the different samples can be obtained. The advantages compared to standard and semi-quantitative PCR techniques are reduction of the assay's time and contamination risks, and improved sensitivity. As for standard PCR, the minimal components of the quantitative PCR reaction mixture are the DNA target of the amplification, an oligonucleotide primer pair flanking the target sequence, a suitable DNA polymerase, deoxynucleotides, buffer and salts. Different technologies have been set up for the monitoring of amplification products, generally based on the use of fluorescent probes. For instance, SYBR Green technology is a non-specific detection system based on a fluorescent dsDNA intercalator and it is applicable to all potential targets. TaqMan technology is more specific since performs the direct assessment of the amount of amplified DNA using a fluorescent probe specific for the target sequence flanked by the primer pair. This probe is an oligonucleotide labelled with a reporter dye (fluorescent) and a quencher (which absorbs the fluorescent signal generated by the reporter). The thermic protocol of amplification allows the binding of the fluorescent probe to the target sequence before the binding of the primers and the starting of the polymerization by Taq polymerase. During polymerization, 5'-3' exonuclease activity of Taq polymerase digests the probe and in this way the reporter dye is released from the probe and a fluorescent signal is detected. The intensity of the signal accumulates at the end of each cycle and is related to the amount of the amplification product. In recent years, quantitative PCR methods based either on SYBR Green or TaqMan technology have been set up for the quantification of Leishmania in mouse liver, mouse skin and human peripheral blood, targeting either single-copy chromosomal or multi-copy minicircle sequences with high sensitivity and reproducibility. In particular, real-time PCR seems to be a reliable, rapid and noninvasive method for the diagnosis and follow up of visceral leishmaniasis in humans. At present, the application of real-time PCR for research and clinical diagnosis of Leishmania infection in dogs is still foreseable. As for standard PCR, the high sensitivity of real-time PCR could allow the use of blood sampling that is less invasive and easily performed for monitoring the status of the dogs. The development of a real-time PCR assay for Leishmania infantum infection in dogs could support the standard and optimized serological and PCR methods currenly in use for the diagnosis and follow-up of canine leishmaniasis, and perhaps prediction of recurrences associated with tissue loads of residual pathogens after treatment. At this regard, a TaqMan Real Time PCR method developed for the quantification of Leishmania infantum minicircle DNA in peripheral blood of naturally infected dogs sampled before and at different time points after the beginning of a standard antileishmanial therapy will be illustrated.     

Non-invasive early prenatal molecular diagnosis using retrieved transcervical trophoblast cells

Fetal DNA was recovered from 17 of 39 (44%) transcervical cell (TCC) samples obtained between 7 and 9 weeks of gestation by endocervical canal flushing. Trophoblast retrieval was adequate for polymerase chain reaction (PCR) amplification of Y chromosome-specific DNA sequences and detection of paternal-specific microsatellite alleles. The fetal sex predicted by PCR in TCCs was confirmed in all cases by karyotype analysis of chorionic villi at 10 weeks of gestation. The absence of the disease-associated paternal alleles in TCC samples from two pregnancies at risk for spinal muscular atrophy and myotonic dystrophy predicted unaffected fetuses in agreement with subsequent results on chorionic villi and newborns' leukocytes. A trisomy 21 fetus was diagnosed in TCCs using fluorescent in situ hybridization (FISH) and semiquantitative PCR analysis of superoxide dismutase-I (SOD 1). Present experience indicates that TCC sampling is a promising technique for early prenatal monitoring of Mendelian disorders and chromosome aneuploidy.     

Detection of male and female fetal DNA in maternal plasma by multiplex fluorescent polymerase chain reaction amplification of short tandem repeats

The purpose of this study was to develop a fluorescent polymerase chain reaction (PCR) assay for the detection of circulating fetal DNA in maternal plasma. Maternal DNA extracted from plasma samples of pregnant women at term and newborn DNA isolated from cord blood were used to genotype 12 mother/child pairs at nine different polymorphic short tandem repeat loci. Multiplex fluorescent PCR was used to detect fetus-specific alleles in the corresponding maternal plasma samples. Fetus-specific alleles were found in all maternal plasma samples studied. Using these polymorphic repeat sequences, every mother/child pair was informative in at least four of nine loci. Paternally inherited fetal alleles were detected in 84% of informative short tandem repeats. This approach may have implications for non-invasive prenatal diagnosis. Compared with other fetal DNA detection systems that use fetus-derived Y sequences to detect only male fetal DNA in maternal plasma, our proposed technique can be applied to both female and male fetuses.     

Rapid detection of trisomies 21 and 18 and sexing by quantitative fluorescent multiplex PCR

Aneuploidies involving chromosomes 21, 18, 13, X and Y account for over 95% of all chromosomal abnormalities in live-born infants. Prenatal diagnosis of these disorders is usually accomplished by cytogenetic analysis of amniotic or chorionic cells but this is a lengthy procedure requiring great technical expertise.In this paper, we assess the diagnostic value of using a quantitative fluorescent polymerase chain reaction (PCR) suitable for the simultaneous and rapid diagnosis of trisomies 21 and 18 together with the detection of DNA sequences derived from the X and Y chromosomes. Samples of DNA, extracted from amniotic fluid, fetal blood or tissues, and peripheral blood from normal adults were investigated by quantitative fluorescent PCR amplification of polymorphic small tandem repeats (STRs) specific for two loci on each of chromosomes 21 and 18. Quantitative analysis of the amplification products allowed the diagnosis of trisomies 21 and 18, while sexing was performed simultaneously using PCR amplification of DNA sequences derived from the chromosomes X and Y. These results indicate the advantages of using two sets of STR markers for the detection of chromosome 21 trisomies and confirmed the usefulness of quantitative fluorescent multiplex PCR for the rapid prenatal diagnosis of selected chromosomal abnormalities. Received: 23 January 1996 / Revised: 21 February 1996     

Endonuclease-mediated long PCR and its application to restriction mapping

The polymerase chain reaction (PCR) is the most widely used technique for the study of DNA. Applications for PCR have been extended significantly by the development of "long" PCR, a technique that makes it possible to amplify DNA fragments up to 40 kb in length. This article describes two novel applications of the long PCR technique, one which simplifies restriction mapping and another which enhances amplification specificity and yield. The same primers used to perform the long PCR amplification can be used as probes to perform restriction mapping of the DNA fragment amplified. Restriction digestion performed prior to long PCR amplification can be used to selectively suppress the amplification of members of families of closely related DNA sequences, thereby making it possible to selectively amplify one of a group of highly homologous sequences. These two complimentary techniques, both involving use of the long PCR paired with restriction digestion, have potential application in any laboratory in which PCR is performed.     

Application of fetal DNA detection in maternal plasma: a prenatal diagnosis unit experience.

Non-invasive prenatal diagnosis tests based on the analysis of fetal DNA in maternal plasma have potential to be a safer alternative to invasive methods. So far, different studies have shown mainly fetal sex, fetal RhD, and quantitative variations of fetal DNA during gestation with fetal chromosomal anomalies or gestations at risk for preeclampsia. The objective of our research was to evaluate the use of fetal DNA in maternal plasma for clinical application. In our study, we have established the methodology needed for the analysis of fetal DNA. Different methods were used, according to the requirements of the assay. We have used quantitative fluorescent polymerase chain reaction (QF-PCR) to perform fetal sex detection with 90% sensitivity. The same technique permitted the detection of fetal DNA from the 10th week of gestation to hours after delivery. We have successfully carried out the diagnosis of two inherited disorders, cystic fibrosis (conventional PCR and restriction analysis) and Huntington disease (QF-PCR). Ninety percent of the cases studied for fetal RhD by real-time PCR were correctly diagnosed. The detection of fetal DNA sequences is a reality and could reduce the risk of invasive techniques for certain fetal disorders in the near future.     

Localised sequence regions possessing high melting temperatures prevent the amplification of a DNA mimic in competitive PCR.

The polymerase chain reaction is an immensely powerful technique for identification and detection purposes. Increasingly, competitive PCR is being used as the basis for quantification. However, sequence length, melting temperature and primary sequence have all been shown to influence the efficiency of amplification in PCR systems and may therefore compromise the required equivalent co-amplification of target and mimic in competitive PCR. The work discussed here not only illustrates the need to balance length and melting temperature when designing a competitive PCR assay, but also emphasises the importance of careful examination of sequences for GC-rich domains and other sequences giving rise to stable secondary structures which could reduce the efficiency of amplification by serving as pause or termination sites. We present data confirming that under particular circumstances such localised sequence, high melting temperature regions can act as permanent termination sites, and offer an explanation for the severity of this effect which results in prevention of amplification of a DNA mimic in competitive PCR. It is also demonstrated that when Taq DNA polymerase is used in the presence of betaine or a proof reading enzyme, the effect may be reduced or eliminated.     

Sex determination in sheep and goats using bovine Y-chromosome specific primers via polymerase chain reaction: potential for embryo sexing.

A simple and novel method, using polymerase chain reaction (PCR) has been standardized for accurate sex determination in sheep and goats. The assay utilizes a pair of bovine Y-chromosome specific primers and the genomic DNA isolated from blood samples of adult male and female sheep and goats. The primers recognize and amplify the Y-chromosome specific sequences in male goats and sheep. The assay is accurate, reliable and rapid.     

Development of a TaqMan quantitative PCR assay specific for Cryptosporidium parvum

A rapid detection method that is both quantitative and specific for the water-borne human parasite Cryptosporidium parvum is reported. Real-time polymerase chain reaction (PCR) combined with fluorescent TaqMan technology was used to develop this sensitive and accurate assay. The selected primer-probe set identified a 138-bp section specific to a C. parvum genomic DNA sequence. The method was optimized on a cloned section of the target DNA sequence, then evaluated on C. parvum oocyst dilutions. Quantification was accomplished by comparing the fluorescence signals obtained from test samples of C. parvum oocysts with those obtained from standard dilutions of C. parvum oocysts. This real-time PCR assay allowed reliable quantification of C. parvum oocysts over six orders of magnitude with a baseline sensitivity of six oocysts in 2 h.     

Prenatal diagnosis of tetrasomy 18p using multiplex fluorescent PCR and comparison with a variety of techniques

We report genetic characterization of isochromosome 18p using a combination of cytogenetic and molecular genetic methods, including multiplex fluorescent PCR. The patient was referred for chorionic villus sampling (CVS) due to advanced maternal age and maternal anxiety. The placental karyotype was 47,XX,+mar, with the marker having the appearance of a small supernumerary isochromosome. Because differentiating between isochromosomes and other structural rearrangements is normally very difficult, a variety of genetic tests including fluorescence in situ hybridization (FISH), PCR, and multiplex fluorescent PCR were undertaken to determine chromosomal origin and copy number and, thus, allow accurate diagnosis of the corresponding syndrome. FISH determined that the marker chromosome contained chromosome 18 material. PCR of a variety of short tandem repeats (STRs) confirmed that there was at least one extra copy of the maternal 18p material. However, neither FISH nor PCR could accurately determine copy number. Multiplex fluorescent PCR (MF-PCR) of STRs simultaneously determined that: (1) the marker included 18p material; (2) the marker was maternal in origin; (3) allele copy number indicated tetrasomy; and (4) contamination of the sample could be ruled out. Results were also rapid with accurate diagnosis of the syndrome tetrasomy 18p possible within 5 hours.     

Sex determination of in vitro developed buffalo (Bubalus bubalis) embryos by DNA amplification

This study was conducted to determine the sex of buffalo embryos produced in vitro by amplifying male specific DNA sequences using the polymerase chain reaction (PCR). This method uses three different pairs of bovine Y-chromosome specific primers and a pair of bovine satellite specific primers. Buffalo in vitro fertilized embryos at the 4-cell to blastocyst stage were collected at days 3, 4, 6, and 8 postinsemination, and the sex of each embryo was determined using all three different Y-chromosome specific primers. The bovine satellite sequence specific primers recognize similar sequences in buffalo and are amplified both in males and in females. Similarly, Y-chromosome specific primers amplify the similar Y-chromosome specific sequences in male embryos of buffalo. Upon examining genomic DNA from lymphocytes of adult males and females, and embryos, the results demonstrate the feasibility of embryo sexing in buffaloes. Furthermore, sex determination by PCR was found to be a rapid and accurate method. © 1993 Wiley-Liss, Inc.     

PCR amplification and analysis of yeast artificial chromosomes

A strategy for the analysis of yeast artificial chromosome (YAC) clones that relies on polymerase chain reaction (PCR) amplification of small restriction fragments from isolated YACs following adapter ligation was developed. Using this method, termed YACadapt, we have amplified several YACs from a human Xq24-qter library and have used the PCR products for physical mapping by somatic cell hybrid deletion analysis and fluorescent in situ hybridization. One YAC, RS46, was mapped to band Xq27.3, near the fragile X mutation. The PCR product is an excellent renewable source of YAC DNA for analyses involving hybridization of YAC inserts to a variety of DNA/RNA sources.     

Fetal gender determination in early first trimester pregnancies of rhesus monkeys (Macaca mulatta) by fluorescent PCR analysis of maternal serum

Non-human primate fetal gender determination can be a powerful tool for research study design and colony management purposes. The recent discovery of the presence of fetal DNA in maternal serum has offered a new non-invasive approach for identification of fetal gender. We present a rapid and simple method for the sexing of developing rhesus monkeys in the first trimester by polymerase chain reaction (PCR) analysis of maternal serum. Serum samples were obtained from 72 gravid rhesus monkeys during 20-32 days of gestation (term 165 +/- 10 days). Fetal gender and the quantity of circulating fetal DNA were determined by real-time PCR analysis of the rhesus Y-chromosomal DNA sequences. The sensitivity for identifying a male fetus was 100% by 30 days gestation, and no false-positive results were observed. This study demonstrates that fetal gender can be reliably determined in the early first trimester from maternal serum samples, a non-invasive method for routine gender screening.     

Analysis of DNA of higher primates using inter-SINE PCR

Two types (MIR and Alu) of short interspersed repeated DNA sequences (SINEs) were used for analysis of genetic relationships among higher primates, and for detection of polymorphism in human genomic DNA. The DNA regions located between the neighboring copies of these SINEs were amplified in polymerase chain reaction with primers complementary to the MIR and Alu consensus sequences (inter-SINE PCR). Comparison of the sets of amplified DNA fragments for different species or individuals provides evaluation of the relationships among them. Using inter-MIR PCR technique, the relationships among the higher primates of the infraorder Catarrhini reported elsewhere were confirmed, pointing to the efficiency of the method for phylogenetic studies. No human DNA polymorphism was revealed with the help of inter-MIR PCR. This polymorphism was detected by means of inter-Alu PCR, which is probably associated with the continuing amplification of Alu elements in human genome.     

Analysis of DNA of higher primates using inter-SINE PCR

Two types (MIR and Alu) of short interspersed repeated DNA sequences (SINEs) were used for analysis of genetic relationships among higher primates, and for detection of polymorphism in human genomic DNA. The DNA regions located between the neighboring copies of these SINEs were amplified in polymerase chain reaction with primers complementary to the MIR and Alu consensus sequences (inter-SINE PCR). Comparison of the sets of amplified DNA fragments for different species or individuals provides evaluation of the relationships among them. Using inter-MIR PCR technique, the relationships among the higher primates of the infraorder Catarrhini reported elsewhere were confirmed, pointing to the efficiency of the method for phylogenetic studies. No human DNA polymorphism was revealed with the help of inter-MIR PCR. This polymorphism was detected by means of inter-Alu PCR, which is probably associated with the continuing amplification of Alu elements in human genome.     

双标准曲线相对定量PCR试验原理与方法

实时荧光定量PCR(FQ-PCR)是一种准确有效的核酸定量分析技术,具有易操作、高通量、高敏感性、高特异性、高度自动化和低污染等优点,并随新定量PCR仪及新操作方法的发展而得到广泛应用,但是,定量PCR的高敏感性特点使得实验操作严格而繁琐。阐述了一种改进的相对定量方法——双标准曲线法的试验原理和特点,描述了定量PCR体系的优化方式,探讨了试验误差分析方法及试验操作技巧,并就试验数据的处理方法进行讨论。试验证明,双标准曲线法是一种经济、简单而准确的定量方法。     

Characterization of TTAGG telomeric repeats,their interstitial occurrence and constitutively active telomerase in the mealybug <Emphasis Type="Italic">Planococcus lilacinus</Emphasis> (Homoptera; Coccoidea)

We confirmed the occurrence of the insect TTAGG telomeric repeats in the mealybug Planococcus lilacinus, a radiation-resistant coccid, by single primer polymerase chain reaction (PCR) and Southern hybridization. Analysis of Bal31 nuclease-digested DNA by Southern hybridization and chromosomes by FISH suggests that these repeats occur mainly at the ends of the chromosomes. However, sequence analysis of the PCR products of TTAGG-associated sequences from genomic DNA showed their interstitial occurrence and association with certain unrelated low-copy repeats. Because of their shorter length, the interstitial TTAGG sequences were detectable by primed in situ hybridizations but not by FISH. Analysis of chromosomes recovered after irradiation by fluorescent in situ hybridization suggested acquisition of TTAGG repeats at a majority of the healed ends. We also observed mild telomerase activity in unirradiated insects which was further enhanced after irradiation. Taken together, these results suggest that the mealybug has an efficient mechanism of formation of TTAGG repeats at radiation-induced chromosome ends and constitutively active telomerase may be a feature associated with rapid recovery of chromosome ends damaged by ionizing radiation.     

Quantitative strain-specific detection of Lactobacillus rhamnosus GG in human faecal samples by real-time PCR

Aims:  To develop a strain-specific rapid assay for identification and quantification of Lactobacillus rhamnosus GG in human faecal samples.
Methods and Results:  A unique random amplified polymorphic DNA (RAPD) band of the L. rhamnosus GG strain was isolated and sequenced. Strain-specific polymerase chain reaction (PCR) primers and probes were designed based on the sequence. Quantification was performed by the real-time PCR using a fluorescent resonance energy transfer (FRET) system. The specificity of the assay was tested with DNA isolated from a set of known strains and human faecal samples. The analytical sensitivity of the method for L. rhamnosus GG was about 10 CFU per assay, which corresponds to 10⁵ CFU g⁻¹ of wet faeces.
Conclusions:  Quantitative real-time PCR is a suitable method for strain-specific identification of L. rhamnosus GG in human faecal samples.
Significance and Impact of the Study:  Lactobacillus rhamnosus GG is one of the most studied probiotic strains in clinical trials but still lacks a DNA-based identification method. This study describes a real-time PCR method for strain-specific identification and quantification of L. rhamnosus GG in human faecal samples.     

Double-step PCR assay to quantify epstein-barr viral load in peripheral blood

Posttransplant lymphoproliferative disorders (PTLD) are a severe complication arising in solid organ transplant patients. A strong correlation between Epstein-Barr virus (EBV) infection, the grade and type of immunosuppression, and the development of PTLD has been recognized. This article describes the development of a double-step polymerase chain reaction (PCR) assay for the quantification of EBV-deoxyribonucleic acid (DNA) to monitor EBV infection. Screening of samples containing >/=10(3) viral genomes/10(5) peripheral blood mononuclear cells (PBMC) or 100 micro L serum by a semiquantitative PCR assay is followed by quantification of the samples containing a high number of viral genomes in a quantitative-competitive (QC)-PCR assay.Screening by semiquantitative PCR selects samples with a high number of viral genomes for use in the more labor-intensive and expensive QC-PCR assay and thus provides a handy means for quantitative DNA analysis of large numbers of samples. Our double-step PCR assay can be employed in EBV viral load measurement in PBMC and serum samples to monitor transplanted patients at risk to develop PTLD.