标本扩增效率的计算及其对HBV DNA定量结果的影响

目的建立分析标本扩增效率的方法，分析其对HBV DNA定量结果的影响。方法应用分析软件提供的样点拟合法及对数荧光一循环数坐标图，计算得到Ct，HBV DNA拷贝数以及每一标本的扩增效率。分析40个HBV DNA拷贝数在10^4-10^8／ml的标本的扩增效率，井用得到的标本实际扩增效率加以校正。结果4个标准的扩增效率分别为0．80，0．80，0．84和0．85。标本的扩增效率在0．59—0．85之问，其中30份（75％）在0．7—0．85之间，8份（20％）在0．65，0．70之问，其余2份在0．65以下。部分校正后的HBV DNA定量结果比校正前高一个数量级。结论标本与标准的扩增效率不一定相同，标本扩增效率的降低导致标准曲线定量结果偏低，有必要用标本的实际扩增效率对结果进行校正。     

Rapid PCR amplification protocols decrease the turn-around time for detection of antibiotic resistance genes in Gram-negative pathogens

A previously designed end-point multiplex PCR assay and singleplex assays used to detect β-lactamase genes were evaluated using rapid PCR amplification methodology. Amplification times were 16–18 minutes with an overall detection time of 1.5 hours. Rapid PCR amplifications could decrease the time required to identify resistance mechanisms in Gram-negative organisms.     

The role of a real-time PCR technology for rapid detection and identification of bacterial and fungal pathogens in whole-blood samples

The rapid diagnosis of pathogens and prompt initiation of appropriate antibiotic therapy are critical factors to reduce the morbidity and mortality associated with sepsis. In this study, we evaluated a multiplex polymerase chain reaction (PCR-M) test that detects bacteria and fungi in whole-blood specimens, comparing its features to those of a blood culture (BC). Following evaluation of the performance for sensitivity and specificity of PCR-M, 78 blood samples from 54 patients with suspected bacterial infections were evaluated. Whole-blood samples for PCR-M were collected at the same time as BC, and PCR-M results were compared with BC results. As a result, minimum sensitivity of the kit was 1–100 cfu/ml. The PCR-M test correctly identified specificity for 13 out of 14 strains blinded to the assay analyst. Of 78 blood samples examined, 56 (72%) were negative by both methods, and 22 (28%) were positive by at least one of the two methods. PCR-M detected organisms in 21 cases (27%) compared with 12 cases (15%) in BC. The correlation of positives between PCR-M and BC was 92% (11/12), and both methods identified the same organisms in these 11 cases. With higher positive rate compared with BC, PCR-M could detect and identify potentially significant microorganisms within a few hours by using a small volume of a single whole-blood sample. Early detection of microorganisms has the potential to facilitate early determination of appropriate treatment and antimicrobial selection.     

Antiprimer quenching-based real-time PCR and its application to the analysis of clinical cancer samples

BACKGROUND: Nucleic acid amplification plays an increasingly important role in genetic analysis of clinical samples, medical diagnostics, and drug discovery. We present a novel quantitative PCR technology that combines the advantages of existing methods and allows versatile and flexible nucleic acid target quantification in clinical samples of widely different origin and quality. METHODS: We modified one of the 2 PCR primers by use of an oligonucleotide "tail" fluorescently labeled at the 5' end. An oligonucleotide complementary to this tail, carrying a 3' quenching molecule (antiprimer), was included in the reaction along with 2 primers. After primer extension, the reaction temperature was lowered such that the antiprimer hybridizes and quenches the fluorescence of the free primer but not the fluorescence of the double-stranded PCR product. The latter provides real-time fluorescent product quantification. This antiprimer-based quantitative real-time PCR method (aQRT-PCR) was used to amplify and quantify minute amounts of input DNA for genes important to cancer. RESULTS: Simplex and multiplex aQRT-PCR demonstrated linear correlation (r(2) >0.995) down to a DNA input equivalent to 20 cells. Multiplex aQRT-PCR reliably identified the HER-2 gene in microdissected breast cancer samples; in formalin-fixed, paraffin-embedded specimens; and in plasma circulating DNA from cancer patients. Adaptation to multiplex single-nucleotide polymorphism detection via allele-specific aQRT-PCR allowed correct identification of apolipoprotein B polymorphisms in 51 of 51 human specimens. CONCLUSION: The simplicity, versatility, reliability, and low cost of aQRT-PCR make it suitable for genetic analysis of clinical specimens.     

The quantification of ADAMTS4 and 8 expression and selection of reference genes for quantitative real-time PCR analysis in myocardial infarction

Introduction

ADAMTS4 and ADAMTS8 are proteases involved in ECM proteolysis and antiangiogenesis, but little is known about their expression and function in myocardial infarction (MI). We examined ADAMTS4 and ADAMTS8 expression in a rat MI model by quantitative real-time polymerase chain reaction (qPCR) and enzyme linked immunosorbent assay (ELISA). The expressions of glyseraldehyde-3-phosphate dehydrogenase (GAPDH), beta-actin (ACTB), acidic ribosomal phosphoprotein P0 (ARBP), and ribosomal protein L13A (RPL13A) were examined in order to validate the appropriate housekeeping genes after MI.

Methods

Male Wistar rats were subjected to MI, and infarcted myocardial tissue was collected at 3, 6, 12, 24 h, 3, 7, 14 and 21 days after MI. ADAMTS4, ADAMTS8, and the four housekeeping genes were quantified using qPCR and the expression stability of the four housekeeping genes was investigated using GeNorm software. The protein levels of ADAMTS4 were detected using ELISA kits.

Results

The M values of GAPDH, ACTB, ARBP and RPL13A were 0.721, 1.2, 0.812 and 0.812 respectively. GAPDH and ARBP were ranked the most stable genes. ADAMTS4 mRNA increased at 3 h after MI, peaked at 6 h, then decreased rapidly. ADAMTS8 mRNA increased at 6 h, peaked at 24 h, remained high at 3 d, then decreased gradually. The protein levels of ADAMTS4 were significantly increased at 6 h, 12 h, 24 h and 3 d after MI.

Conclusion

The results suggest that GAPDH and ARBP are two appropriate housekeeping genes for the rat MI model. Both ADAMTS4 and ADAMTS8 mRNA levels and ADAMTS4 protein level increased, but they exhibited different expression profiles.     

HBV-HCV-HIV三联荧光PCR检测的内标浓度选择研究

目的选择一个在乙型肝炎病毒-丙型肝炎病毒-人类免疫缺陷病毒(HBV-HCV-HIV)三联荧光PCR检测试验中既可有效监控假阴性结果出现,又对阳性结果影响最小的内标浓度。方法应用不同浓度的内标参入酶链聚合反应(PCR)反应,确定最佳内标参入量;并在适量内标浓度下,检测低浓度标本的阳性检出率。结果由内标浓度5拷贝/PCR、10拷贝/PCR、20拷贝/PCR、50拷贝/PCR、100拷贝/PCR 5个浓度中,优选出最适的内标浓度为20拷贝/PCR,此条件下,阴性标本的内标检出率为100%,检测灵敏度标本的阳性检出率与无内标样本差异无统计学意义。结论合适浓度的内标参与荧光PCR检测能有效地解决了每个标本的质控问题,指示反应体系(试剂耗材)与检测体系(仪器)的有效性。     

HBV-HCV-HIV三联荧光PCR检测的内标浓度选择研究

目的 选择一个在乙型肝炎病毒-丙型肝炎病毒-人类免疫缺陷病毒（HBV—HCV—HIV）三联荧光PCR检测试验中既可有效监控假阴性结果出现,又对阳性结果影响最小的内标浓度。方法应用不同浓度的内标参入酶链聚合反应（PCR）反应,确定最佳内标参人量;并在适量内标浓度下,检测低浓度标本的阳性检出率。结果由内标浓度5拷贝／PCR、10拷贝／PCR、20拷贝／PCR、50拷贝／PCR、100拷／PCR5个浓度中,优选出最适的内标浓度为20拷贝／PCR,此条件下,阴性标本的内标检出率为100％,检测灵敏度标本的阳性检出率与无内标样本差异无统计学意义。结论合适浓度的内标参与荧光PCR检测能有效地解决了每个标本的质控问题,指示反应体系（试剂耗材）与检测体系（仪器）的有效性。     

Detection of Neisseria Meningitidis in clinical samples by a duplex real-time PCR targeting the porA and ctrA genes.

BACKGROUND: In recent years PCR has proven to be a highly sensitive and specific method for the diagnosis of infections caused by Neisseria meningitidis. STUDY DESIGN: We developed and evaluated a N. meningitidis LightCycler real-time duplex PCR (NM-LCdPCR) capable of simultaneously detecting and distinguishing between two separate genes on the N. meningitidis genome. METHODS: The NM-LCdPCR was developed on the LightCycler platform (Roche Diagnostics, Castle Hill, NSW, Australia) and comprised two primer pairs and two hybridization probe sets, enabling the detection of both the porA and ctrA genes within the same reaction mix. To distinguish between the fluorescence emitted by each hybridization probe set, each downstream probe was labeled with a different fluorophore (either LC-Red640 or LC-Red705). The results obtained by the NM-LCdPCR were then compared with the results obtained by a mono-specific LightCycler assay targeting the porA gene only (porA-LCPCR). PATIENTS: One-hundred and forty-eight clinical samples from patients with suspected meningococcal infection were evaluated. RESULTS: The results of the NM-LCdPCR and porA-LCPCR gave 100% agreement; N. meningitidis DNA was detected in 25 samples whereas 123 samples were negative by both assays. The breakdown of the NM-LCdPCR results show that both genes were detected in 26 of the 28 positive samples. DISCUSSION: By targeting two separate N. meningitidis genes, the NM-LCdPCR has the potential to prevent the false-positive results which may arise from sequence variation. In addition, the ability to detect and discriminate between the two different N. meningitidis genes within the same reaction mix offers a rapid means for confirming the presence of N. meningitidis DNA in clinical samples, thereby reducing the need for subsequent confirmatory assays to be performed. CONCLUSIONS: The sensitivity and specificity of the NM-LCdPCR assay, combined with its ability to detect and discriminate both the N. meningitidis porA and ctrA genes, make it suitable for the diagnosis of N. meningitidis infections in the routine clinical laboratory.     

HER-2 DNA quantification of paraffin-embedded breast carcinomas with LightCycler real-time PCR in comparison to immunohistochemistry and chromogenic in situ hybridization

OBJECTIVES: To compare the detection of HER-2 status by real-time PCR, on paraffin-embedded breast carcinomas, in respect to immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH). DESIGN AND METHODS: Paraffin-embedded breast carcinomas collected from 85 patients diagnosed with early stage breast cancer were analyzed for HER-2 gene amplification by real-time PCR and CISH, as well as for HER-2 protein expression by IHC. RESULTS: HER-2 gene amplification was observed in 19 (22.4%) of 85 breast cancer patients by real-time PCR and in 19 (22.4%) of 85 patients by CISH. Strong (3+) HER-2 protein over-expression was observed in 13 (15.3%) out of 85 patients. Moreover, there were 4 out of 85 (4.7%) patients that had moderate (2+) HER-2 protein over-expression, while 68 out of 85 (80%) patients had no HER-2 protein over-expression by IHC. There were strong concordance rates between real-time PCR and IHC (79/85, 92.9%, p<0.0001) and real-time PCR and CISH (77/85, 90.6%, p<0.0001). The concordance rate between the three methods was 90.6% (p<0.0001). CONCLUSIONS: Our data show that the results obtained for amplification of HER-2 by real-time PCR on the LightCycler are comparable to those obtained by IHC and CISH.     

Detection and genotyping by real-time PCR of the staphylococcal enterotoxin genes sea to sej

This paper describes real-time fluorescence PCR assays for detecting and toxinotyping nine enterotoxin genes from Staphylococcus aureus. A universal set of primers allowed sea, seb, sec, sed, see, seg, seh, sei, sej enterotoxin genes from S. aureus to be detected in a single real-time PCR assay with the LightCycler (LC) instrument. Using the universal forward primer and a type-specific reverse primer, real-time PCR assays allowed the S. aureus enterotoxin genes to be specifically genotyped. A collection of S. aureus isolates (n=83) was detected and further characterised for sea, seb, sec, sed, see, seg, seh, sei, sej, using real-time PCR assays, and data were compared with those obtained by conventional block cycler PCR. Isolates were also tested for their ability to produce staphylococcal enterotoxins A, B, C and D by a commercial reversed passive latex agglutination (RPLA) test. Real-time PCR assays developed on the LightCycler system (LC-PCR) are a powerful tool for rapid detection and toxinotyping of the enterotoxin genes sea to sej from S. aureus. The work offers a very quick, reliable and specific alternative to conventional block cycler PCR assays to identify the enterotoxin profile of toxigenic S. aureus.     

Real-time PCR method with statistical analysis to compare the potential of DNA isolation methods to remove PCR inhibitors from samples for diagnostic PCR

A real-time PCR method for fast comparison of different DNA isolation methods to remove PCR inhibitors from samples is presented. A fixed amount of target-200 copies of a 79-bp region of the COCH gene of the zebrafish (Danio rerio)-was added to each PCR reaction together with isolated DNA from different types of samples including chicken feces. Four commercial DNA isolation kits and a chelex-based technique were compared using this method. The copy numbers calculated and the endpoint fluorescence were statistically compared to the values of 22 control samples containing the control target and water instead of isolated DNA, processed together in the same PCR run. The level of the endpoint fluorescence was more often negatively influenced by inhibitors than the copy number calculated, suggesting a more pronounced effect on the plateau phase of the reaction by limiting one or more compounds in the PCR reaction.     

PCR and sequencing of independent genetic targets for the diagnosis of culture negative bacterial endocarditis

Molecular methods utilizing broad-range primers for 16S rDNA PCR and sequencing have been widely evaluated for their utility in culture negative diagnostic bacteriology. Difficulties in determining the incidence of false positive PCR results, especially in the absence of an equally sensitive confirmatory method however, have prevented wide clinical use of this sensitive technology. Here we report two cases of culture-negative endocarditis, in which PCR using 16S rDNA broad-range primers generated sequences specific for Bartonella spp. and Streptococcus oralis, respectively. To confirm these results, a second species- or genus-specific molecular target was chosen for each organism and detected in the split samples sequencially. Thus, molecular detection of a second species-specific target can be used to confirm PCR results generated from 16S rDNA broad-range primers and to control for potential false positive results due to environmental and amplicon carry-over contamination during specimen processing and testing in the laboratory.     

Ecology and evolution as targets: the need for novel eco-evo drugs and strategies to fight antibiotic resistance

In recent years, the explosive spread of antibiotic resistance determinants among pathogenic, commensal, and environmental bacteria has reached a global dimension. Classical measures trying to contain or slow locally the progress of antibiotic resistance in patients on the basis of better antibiotic prescribing policies have clearly become insufficient at the global level. Urgent measures are needed to directly confront the processes influencing antibiotic resistance pollution in the microbiosphere. Recent interdisciplinary research indicates that new eco-evo drugs and strategies, which take ecology and evolution into account, have a promising role in resistance prevention, decontamination, and the eventual restoration of antibiotic susceptibility. This minireview summarizes what is known and what should be further investigated to find drugs and strategies aiming to counteract the "four P's," penetration, promiscuity, plasticity, and persistence of rapidly spreading bacterial clones, mobile genetic elements, or resistance genes. The term "drug" is used in this eco-evo perspective as a tool to fight resistance that is able to prevent, cure, or decrease potential damage caused by antibiotic resistance, not necessarily only at the individual level (the patient) but also at the ecological and evolutionary levels. This view offers a wealth of research opportunities for science and technology and also represents a large adaptive challenge for regulatory agencies and public health officers. Eco-evo drugs and interventions constitute a new avenue for research that might influence not only antibiotic resistance but the maintenance of a healthy interaction between humans and microbial systems in a rapidly changing biosphere.     

Evaluation of a multiplex PCR assay for simultaneous detection of bacterial and viral enteropathogens in stool samples of paediatric patients

We evaluated a multiplex PCR assay, the Seeplex Diarrhoea ACE detection, that simultaneously detects 15 enteric pathogens, including Salmonella spp., Shigella spp., Vibrio spp., toxin B producer Clostridium difficile, Campylobacter spp., Clostridium perfringens, Yersinia enterocolitica, Aeromonas spp., Escherichia coli O157:H7, verocytotoxin-producing Escherichia coli, adenovirus, Group A rotavirus, norovirus GI and GII, and astrovirus. We compared this assay with clinical methods routinely used in our laboratory, for detecting enteropathogens in stool samples collected from 245 paediatric patients with suspected infectious gastroenteritis. We recovered 61 bacterial pathogens and 121 enteric viruses with our laboratory assays, while we detected 78 bacteria and 167 viruses with the molecular assay. We calculated specificity and sensitivity for both methods after analysis of discordant results and demonstrated greater sensitivity for multiplex PCR than for our routine methods, with the exception of Salmonella spp. and toxigenic C. difficile detection. The multiplex PCR assay proved to be a reliable tool to directly detect the most common enteropathogens in stool samples but with some limitations.     

Evaluation of the RIDAGENE real-time PCR assay for the detection of GI and GII norovirus

The current study examined the efficacy of the RIDAGENE norovirus (NoV) real-time polymerase chain reaction assay (R-Biopharm, Darmstadt, Germany) for use in a routine diagnostic laboratory. The RIDAGENE assay had an overall sensitivity of 98% but was more sensitive for GII than GI NoV. The assay had a specificity of 98%. The RIDAGENE assay could detect a variety of GI and GII open reading frame 2 genotypes including GI.1, GI.3, GI.8, GI.13, GII.2, GII.3, GII.4 (including the following variants: 2006b, 2009, 2012, and 3 others that have not been assigned), GII.6, GII.12, and GII.13. The assay did not cross react with a number of gastroenteritis viruses including adenovirus, astrovirus, rotavirus, and sapovirus. The assay was straightforward to perform, and for a run of 50 specimens, a result was obtainable in roughly 4 hours. The RIDAGENE assay can be recommended as a valuable detection method for NoV.     

Rapid real-time fluorescent PCR gene dosage test for the diagnosis of DNA duplications and deletions

BACKGROUND: Current methods to determine gene dosage are time-consuming and labor-intensive. We describe a new and rapid method to assess gene copy number for identification of DNA duplications or deletions occurring in Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP), respectively. METHODS: We studied 16 patients with HNPP, 4 with CMT1A, and 49 control subjects. We used real-time PCR on the LightCycler system with use of a single capillary tube and no post-PCR handling. A polymorphic fragment of the PMP22 gene was amplified to determine gene dosage for heterozygous samples. The presence of two alleles was used to indicate that no deletion was present in HNPP samples. The ratio obtained between the areas under each allele melting curve of heterozygous CMT1A samples was used to determine whether the sequence was duplicated or normal. Homozygous samples required a competitive gene dosage test, where the ratio between the areas under the melting curves of the target DNA of samples and of the competitor molecule was used to determine whether the target sequence was duplicated, deleted, or normal. Samples from HNPP, CMT1A, and controls were analyzed. RESULTS: Area ratios were approximately 0.6, 1.0, and 2.0 for HNPP, control, and CMT1A samples, respectively. The results agreed with those obtained by Southern blotting and microsatellite analysis in the same samples. CONCLUSIONS: Direct and competitive real-time fluorescent PCR can differentiate one, two, or three copies of the target DNA. The method described is sensitive and accurate for detection of CMT1A duplications and HNPP deletions and is faster and easier than current methods.     

Evaluation of an extended diagnostic PCR assay for detection and verification of the common causes of bacterial meningitis in CSF and other biological samples

A seminested polymerase chain reaction (PCR)-based diagnostic assay was evaluated for detection and verification of Neisseria meningitidis, Haemophilus influenzae, Streptococcus pneumoniae, Steptococcus agalactiae and Listeria monocytogenes in cerebrospinal fluid (CSF) and other biological samples. A general bacterial amplicon from the 16S rRNA gene was amplified in a first step, and species-specific regions in a second. The detection level was 4 fg DNA/reaction, corresponding to about one bacterial genome per reaction tube. Sample preparations (Dynabeads DNA DIRECT kit) were assayed from 140 bacterial strains suspended in saline. In CSF the detection level for bacteria was 10(3)CFU ml-1for N. meningitidis, H. influenzae and S. pneumoniae, 10(4)CFU ml-1for Escherichia coli and 10(5)CFU ml-1for S. agalactiae and L. monocytogenes. The detection levels for these bacteria were the same in the other tested biological samples, like blood with or without culture media. Clinical CSF samples were evaluated from 71 patients with proven bacterial meningitis, as were 61 CSF samples from individuals without bacterial meningitis. The diagnostic sensitivity of the assay in detecting bacteria in general was 0.97, and for the specific species in the clinical CSF samples 0.87-0.94. The specificity was 1.0 for detecting bacteria in general. Some cross-reactions were noted within the streptococcus group. The PCR results were verified by banding patterns of Hae III digested PCR products.