A comparative evaluation of the sensitivity of two automated and two manual nucleic acid extraction methods for the detection of norovirus by RT-PCR

The aim of the study was to compare the sensitivity of a norovirus RT-PCR method using two manual RNA extraction methods (Qiagen and Roche) and two automated RNA extraction methods (Qiagen and Corbett). All four RNA extraction methods gave similar sensitivities although the automated methods, especially the Corbett, required significantly less labour than the manual methods. The automated methods also enabled RNA extraction of approximately two to three times the number of specimens in a given time period compared to manual methods.     

Evaluation of automated nucleic acid extraction methods for virus detection in a multicenter comparative trial

Five European veterinary laboratories participated in an exercise to compare the performance of nucleic acid extraction robots. Identical sets of coded samples were prepared using serial dilutions of bovine viral diarrhoea virus (BVDV) from serum and cell culture propagated material. Each laboratory extracted nucleic acid from this panel using available robotic equipment (12 separate instruments, comprising 8 different models), after which the processed samples were frozen and sent to a single laboratory for subsequent testing by real-time RT-PCR. In general, there was good concordance between the results obtained for the different automated extraction platforms. In particular, the limit of detection was identical for 9/12 and 8/12 best performing robots (using dilutions of BVDV infected-serum and cell culture material, respectively), which was similar to a manual extraction method used for comparison. The remaining equipment and protocols used were less sensitive, in an extreme case for serum, by a factor of 1000. There was no evidence for cross-contamination of RNA template in any of the negative samples included in these panels. These results are not intended to replace local optimisation and validation, but provide reassurance to laboratories to indicate that the best performing optimised nucleic acid extraction systems can have similar performance.     

Comparison of automated and manual methods for urinalysis

The authors compared results for accuracy and precision obtained by a semiautomated prototype International Remote Imaging Systems, Inc. (IRIS) urinalysis workstation (IUW) with those from quantitative manual urinalysis (QMU). Three technologists skilled in urinalysis each performed 172 urinalyses with both the IUW and QMU methods. The results show that the IUW method is likely to yield comparable counts for particulate analytes compared with the QMU, except for casts. The QMU reported significantly (P less than 0.001) more casts than the IUW method. This difference is related to at least a ninefold greater volume of untreated urine examined by the QMU method than the IUW method. The IUW method may provide a more accurate result than the QMU method at very low and high concentrations of particulate analytes. The result from 24 blind duplicate urines also analyzed by each of the three technologists with both methods showed comparable precision for particulate analytes between the two methods except for red blood cells; the QMU method had significantly (P less than 0.001) better precision for this analyte.     

Rapid detection of enterovirus infection by automated RNA extraction and real-time fluorescence PCR.

BACKGROUND: Molecular detection has been shown to be superior to tissue culture for the detection of enteroviruses in cerebrospinal fluid (CSF) specimens. OBJECTIVES: In this study, a qualitative molecular assay based on automated RNA extraction with the MagNA Pure LC and real-time PCR on the LightCycler (LC) instrument was evaluated and compared with an in-house molecular assay. STUDY DESIGN: A total of 109 CSF specimens were investigated for the comparative study. The detection limit of the new molecular assay was determined with 10-fold dilutions of two enterovirus strains and with the Third European Union Concerted Action Enterovirus Proficiency Panel. RESULTS: With the enterovirus strains, the detection limit of the LC assay was found to be 0.1 TCID(50) (50% tissue culture infective dose). When samples of the Third European Union Concerted Action Enterovirus Proficiency Panel were tested, both molecular assays gave identical results to the expected results, which were based upon the results of three reference laboratories using a total of four different molecular methods before distribution of the panel. When clinical specimens were tested, there was a correlation between the LC assay and the in-house assay in 105 of 109 cerebrospinal fluids. CONCLUSIONS: The new molecular assay allows rapid detection of enterovirus RNA in CSF. It was found to be labor saving and showed sufficient sensitivity.     

Rapid enterovirus RNA detection in clinical specimens by using nucleic acid sequence-based amplification

Enterovirus (EV) detection by nucleic acid sequence-based amplification was compared with EV isolation in cell culture. The NucliSens Basic kit (bioMerieux) was utilized for RNA detection. For virus isolation, samples were inoculated into MRC-5, primary rhesus monkey kidney, A549, rhabdomyosarcoma, and/or Buffalo green monkey kidney cells.     

Comparison of six nucleic acid extraction methods for detection of viral DNA or RNA sequences in four different non-serum specimen types.

BACKGROUND: Extraction of viral nucleic acids from serum samples is widely used in diagnostic pathology tests. However, the heterogeneous nature of non-serum samples may contribute to variations in the yields of viral nucleic acids with different extraction methods and specimen types. OBJECTIVES: Six different methods were compared for optimal extraction of viral DNA or RNA from four types of non-serum specimens. STUDY DESIGN: The DNA viruses used were herpes simplex virus and cytomegalovirus. The RNA viruses were poliovirus, rotavirus and small round structured virus. The specimens used were from respiratory, genital, faecal and peripheral blood mononuclear cell samples. The extracted nucleic acids were amplified by PCR and detected in an enzyme immunoassay using digoxygenin-labelled amplicons. RESULTS AND CONCLUSIONS: For extraction of viral DNA, the phenol-chloroform method yielded the highest amount of DNA as judged by endpoint titration. The three methods compared for extraction of viral RNA used guanidine isothiocyanate and the QiaRNA kit was shown to yield the highest amount of viral RNA.     

Real-time nucleic acid sequence-based amplification using molecular beacons for detection of enterovirus RNA in clinical specimens

Real-time nucleic acid sequence-based amplification (NASBA) using molecular beacon technology (NASBA-beacon) was compared to standard NASBA with postamplification hybridization using electrochemiluminescently labeled probes (NASBA-ECL) for detection of enteroviruses (EV) in 133 cerebrospinal fluid and 27 stool samples. NASBA-ECL and NASBA-beacon were similar in sensitivity, detecting 55 (100%) and 52 (94.5%) EV-positive samples, respectively. There were no false positives. Both NASBA assays were significantly more sensitive than culture. Real-time NASBA-beacon reagents and equipment rental were more expensive than those for NASBA-ECL; however, time to result was shortened by 1.5 h, hands-on time was reduced by 25 min, and the assay was much simpler for technologists to learn and perform.     

Comparison of automated and rapid manual methods for the same-day identification of Enterobacteriaceae

The Vitek AMS automated instrument method for identification of Enterobacteriaceae was compared with two rapid manual methods intended for the same purpose, the Micro ID System and the API 20E Same-Day procedure, on a series of 400 consecutive fresh clinical isolates. Results were compared with identifications obtained using the API 20E System with overnight incubation and supplemental tube biochemicals (when needed). Both the final (8-hour) and a manually requested, presumptive 5-hour result from the AMS were compared with the 4-hour results provided by the Micro ID and the 5-hour results provided by the API. The Micro ID system proved to be the most rapid and accurate of the three test systems by correctly identifying 96.8% (387/400) of isolates. The API 20E using 5-hour readings identified 90.7% (363/400) of isolates, although 96.8% (387/400) could be identified if supplemental overnight tests were employed to separate profile codes with "good likelihood, but low selectivity." The AMS correctly identified 88.8% (355/400) isolates after 5 hours, and 95.0% (380/400) following 8 hours incubation.     

Viral nucleic acid stabilization by RNA extraction reagent

In the collection of field materials to test for the presence of arboviruses, samples must be appropriately maintained to detect arboviral nucleic acids. In austere field conditions this is often difficult to achieve because, during routine specimen processing, storage, and shipping viral RNA degradation could result in detection failure. RNA extraction reagents, while used commonly for their intended purpose of stabilizing RNA during the extraction process, have not been assessed fully for their potential to stabilize RNA before extraction. The potential for virus stabilization at varying temperatures and periods of time remains unknown. Accordingly, the ability of buffer AVL (Qiagen, Valencia, CA), an RNA extraction reagent, to stabilize viral suspensions of dengue, Venezuelan equine encephalitis and Rift Valley fever viruses was evaluated. The ability of buffer AVL to stabilize each viral suspension was examined at 32, 20, 4, and −20 °C. RNA in samples placed in buffer AVL was stable for at least 48 h at 32 °C and refrigerating samples prolonged stabilization. Additionally, placing the sample/buffer AVL mixture at either 4 or −20 °C stabilized samples for at least 35 days. When combined with the ability of buffer AVL to inactivate viral samples, this provides the ability to collect and handle potentially infectious samples in a safe way that also provides sample stabilization.     

应用核酸分子杂交技术检测心肌炎组织中的肠道病毒RNA

用生物素标记ｐＣＢＩＩＩ／３５和ｐＣＢＩＩＩ／５１肠道病毒属特异性探针，对４５心肌炎和心肮病尸检或心肌活检心脏组织进行原位杂交，结果显示：１２例（２６．６％）存在肠道病毒ＲＮＡ。柯萨基Ｂ３病毒型特异性探针ｐＣＢＩＩＩ／２９进一步杂交表明，１２例经证实存在肠道病毒ＲＮＡ的心肌组织中５例（４１．７％）出现阳性杂交信号。阳性杂信号主要位于单个或多个心肌细胞的胞浆和心肌的间质组织，偶见竽心肌小血管内皮细胞     

Evaluation of NucliSens easyMAG for automated nucleic acid extraction from various clinical specimens

The objectives of this study were to evaluate the performance of the NucliSens easyMAG platform for nucleic acid extraction from different clinical specimens compared to NucliSens miniMAG platform and manual QIAGEN extraction. The NucliSens easyMAG and the NucliSens miniMAG showed equal performance on 215 throat swabs since real-time nucleic acid sequence-based amplification scored the same samples positive for Mycoplasma pneumoniae (n=9) and Chlamydia pneumoniae (n=5) RNAs, although internal control RNA was slightly better detected with the NucliSens easyMAG (99.3% versus 96.8%). NucliSens easyMAG extracted nucleic acids more efficiently (higher recovery and/or fewer inhibitors) compared to QIAGEN extraction by showing, on average, lower Ct values in real-time LightCycler PCR, although 4 individual specimen out of 45 were found positive only with QIAGEN. For nine M. pneumoniae-positive throat swabs, the mean difference in Ct values between NucliSens easyMAG extraction and QIAGEN extraction was -2.26 (range, -5.77 to +0.60); for the detection of five C. pneumoniae-positive throat swabs, the average difference in Ct values between the two methods was -3.38 (range, -6.62 to -2.02); and for the detection of cytomegalovirus in 24 blood samples, the mean difference in Ct values between the two methods was -0.95 (range, -5.51 to +1.68). The NucliSens easyMAG is considerably easier to perform, efficiently extracts nucleic acids from throat swabs and whole blood, is automated, and has high throughput.     

Comparison of the NucliSens easyMAG and Qiagen BioRobot 9604 nucleic acid extraction systems for detection of RNA and DNA respiratory viruses in nasopharyngeal aspirate samples

The NucliSens easyMAG and BioRot 9604 automated nucleic acid extraction systems were evaluated and compared with the manual QIAamp (Qiagen) extraction method for their abilities to extract nucleic acid from nasopharyngeal aspirate samples for the detection of RNA and DNA respiratory viruses. The nucleic acids recovered by all three methods gave comparable sensitivities in PCR tests, and the three methods gave comparable viral loads. There was no evidence of residual PCR inhibitors and no evidence of PCR cross-contamination.     

Direct and fast detection of methicillin resistant Staphylococcus aureus carriage by automated nucleic acid extraction and real time PCR

We have developed a real time PCR assay for methicillin resistant Staphylococcus aureus (MRSA) screening able to provide a result in less than 3 h. The PCR amplifies a 184 bp fragment corresponding to the junction area between mecA and orfX genes that allows specific identification of MRSA in a nonsterile specimen. 1481 nasal swabs taken from geriatrics, dialysis and intensive care patients were compared with traditional bacteriology. A short centrifugation, preliminary to the extraction, with "SETS" system allows a recovery of the sample. The automated DNA extraction is carried out by the MagNA Pure LC and the PCR by the LightCycler. The agreement between the two methods is 97.7%. A study of sensitivity and specificity on 1111 samples respectively gives 75 and 98% for the real time PCR and, 64 and 99% for the culture. The strategy of fast and effective tracking that we propose is of an undeniable contribution in the fight against the MRSA infections.     

磁珠法提取核酸检测丙肝病毒RNA载量及其临床研究

目的 探讨磁珠法提取核酸检测丙肝病毒RNA载量的方法学评价及其临床应用.方法 采用精密度、检测下限、线性分析和干扰实验对磁珠法提取核酸进行方法学评价,采用磁珠法和柱抽提法对283份标本进行检测,进行相关性分析.结果 磁珠法的高值质控血清的批内精密度为6.64±0.11,CV为1.65％,批间精密度为6.68±0.14,CV为2.09％;低值质控血清的批内精密度为4.60±0.12,CV为2.66％,批间精密度为4.56±0.19,CV为4.10％;检测下限为用磁珠法检测20份500IU/L HCV血清标本,20份标本均扩增出曲线,阳性率为100％（20/20）.磁珠法线性评估结果呈明显线性,相关系数r=0.999,P＜0.05,回归方程y=0.982x＋ 0.092.磁珠法和柱抽提法提取核酸检测HCV-RNA载量的比较,磁珠法与柱抽提法的相关系数r=0.990,P＜0.05,回归方程y=0.965x ＋0.247.结论 磁珠法提取核酸检测丙肝病毒RNA载量的方法具有较高的特异性和灵敏度,较强的抗干扰能力,且方法简便易于自动化操作,是理想的实验室核酸提取方法.     

Comparison of RNA extraction methods for the detection of porcine reproductive and respiratory syndrome virus from boar semen

To detect Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) in semen, various RNA extraction techniques have been utilized for RT-PCR, but rarely compared, to determine an optimized extraction protocol. Due to the viscosity, non-homogeneity, high cellularity and large volume of boar semen produced, difficulties can be encountered in obtaining RNA from the seminal cell fraction. This study compared six RNA extractions, five which used a commercially available kit (RNeasy, Qiagen Inc.) for use on highly cellular samples and a traditional phenol/chloroform procedure. All extractions were compared on serially diluted PRRSV "spiked" seminal cell fractions. The two methods resulting in recovery of the highest amount of RNA, which included a Qiashredder (Qiagen Inc.) (protocol 1) or cell lysis/centrifugation technique (protocol 3) preceding the RNeasy procedure were then compared using naturally infected semen samples from experimentally infected boars. Both protocols detected similar amounts of virus in "spiked" samples, but protocol 1 detected eight additional PRRSV-positive semen samples in naturally infected semen. This study demonstrated that semen "spiked" with PRRSV (cell-free virus) may not be representative of naturally infected semen samples (cell associated virus) for comparing extraction protocols, but did identify a useful extraction technique for boar semen.     

Cytomegalovirus DNA quantification using an automated platform for nucleic acid extraction and real-time PCR assay setup

Analytical performance characteristics of the QIAsymphony RGQ system with artus cytomegalovirus (CMV) reagents were determined. Measurable range spanned 2.0 to ≥ 7.0 log(10) copies/ml. The detection limit was 23 copies/ml. Intrarun and interrun coefficients of variation were ≤ 2.1% at 3.0 and 5.0 log(10) copies/ml. In clinical specimens, RGQ values were ~0.2 log(10) copies/ml higher than those in an assay using a BioRobot M48 extraction/manual reaction setup/7500 Real-Time PCR instrument. No cross-contamination was observed.