Use of a Liposomal Internal Control Vehicle for Whole-Process Quality Assurance of Nucleic Acid Amplification

2013 ◽  
pp. 19-36
Author(s):  
Einar Berg ◽  
Tania Nolan
Keyword(s):  
Quality Assurance ◽  
Nucleic Acid ◽  
Internal Control ◽  
Process Quality ◽  
Nucleic Acid Amplification ◽  
Whole Process

Empfehlungen zur qualitätsgesicherten Durchführung von Nukleinsäure-Amplifikations - Testungen (NAT) von Blutprodukten. Quality assurance in nucleic acid amplification testing (NAT) of blood products

2005 ◽  
Vol 29 (2) ◽  
Author(s):  
Willi K. Roth
Keyword(s):  
Quality Control ◽  
Nucleic Acid ◽  
Sample Preparation ◽  
Nucleic Acids ◽  
Internal Control ◽  
Control Measures ◽  
Nucleic Acid Amplification ◽  
Blood Products ◽  
Whole Process ◽  
And Storage

AbstractEuropean manufacturers of plasma products and German blood transfusion services were the first to introduce nucleic acid amplification testing (NAT) of blood products in the mid-1990s. Their primary goal was to increase the safety of blood by closing as far as possible the diagnostic window, which exists after the onset of viral infection until the appearance of the first detectable antibodies. Sample preparation, transport and storage are crucial steps in a quality-controlled PCR. Sensitivity and contamination rates highly depend on the sample preparation and storage techniques. Anticoagulants must be selected carefully because some may inhibit the PCR. Dilution of samples by pooling needs to be considered and should be compensated for by subsequent virus enrichment procedures, e.g. centrifugation. The whole process of sample preparation, pooling and virus enrichment must be validated and quality control measures must be implemented. Reagents for the extraction of viral nucleic acids should not pose any risk to the laboratory staff. Nevertheless, the reagents should be highly efficient in liberating viral nucleic acids at high yield and purity for the following amplification reactions. At this critical stage, quality control measures should guarantee an efficient extraction process and contain potential sources of contaminations. Several methods are available for the amplification of nucleic acids. PCR is the most common, especially in in-house assays. The amplification of nucleic acids should be performed as far as possible in a closed system, which may be guaranteed best by real-time PCR approaches. Reaction tubes need never be opened during the amplification because detection can be performed through the closed tube. Amplicons that could contaminate the following PCR reactions will not be released. It is of great importance to blood transfusion services to guarantee that negative results un-equivocally indicate virus negative blood donations. Therefore, internal control sequences should be implemented in each individual PCR reaction in order to monitor that the individual PCR has worked correctly. Besides internal control sequences, external negative and positive controls should be implemented in each PCR run to demonstrate false positive reactions as well as to monitor pre-PCR processes like virus enrichment and extraction. The whole process needs to be validated according to the criteria set in national guidelines or by national authorities. External quality assessment programs are highly recommended.

Inhibition of nucleic acid amplification assays: is an internal control necessary?

1996 ◽  
Vol 27 (1) ◽  
pp. 97-98 ◽  
Author(s):  
G. Leckie ◽  
J. Cao ◽  
Q. He ◽  
D. Kawa ◽  
D. Erickson ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Internal Control ◽  
Nucleic Acid Amplification

scholarly journals Evaluation of AMPLILINK Software for the COBAS AMPLICOR System

1999 ◽  
Vol 37 (2) ◽  
pp. 436-437 ◽  
Author(s):  
Harald H. Kessler ◽  
Donald Jungkind ◽  
Evelyn Stelzl ◽  
Sue Direnzo ◽  
Srinivas K. Vellimedu ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Internal Control ◽  
Diagnostic Procedures ◽  
Nucleic Acid Amplification ◽  
Molecular Diagnostic ◽  
Single Specimen ◽  
Clinical Laboratories ◽  
And Control

The use of AMPLILINK version 1.0 software was evaluated for the operation and control of one COBAS AMPLICOR instrument and for two COBAS AMPLICOR instruments run simultaneously to perform and detect nucleic acid amplification reactions. A total of 3,384 results were analyzed. The initial accuracy of the results was 99.91%. Three errors of omission of transfer of data from the COBAS AMPLICOR to the AMPLILINK system were observed. Two of these errors were from a single specimen, where both the analyte and internal control results were not transmitted. These errors did not interfere with the correctness of any other data. There were no interruptions of runs, and no data were mixed. AMPLILINK increased convenience, saved labor, and was found to be a very useful addition for clinical laboratories performing molecular-diagnostic procedures with the COBAS AMPLICOR system.

HIV Detection from Human Serum with Paper-Based Isotachophoretic RNA Extraction and Reverse Transcription Recombinase Polymerase Amplification

2021 ◽  
Author(s):  
Andrew Bender ◽  
Benjamin Sullivan ◽  
Jane Zhang ◽  
David Juergens ◽  
Lorraine Lillis ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Human Serum ◽  
Internal Control ◽  
Reverse Transcription ◽  
Low Cost ◽  
Rna Extraction ◽  
Recombinase Polymerase Amplification ◽  
Nucleic Acid Amplification ◽  
People Living With Hiv ◽  
Ms2 Bacteriophage

<p>The number of people living with HIV continues to increase with the current total near 38 million, of which about 26 million are receiving antiretroviral therapy. These treatment regimens are highly effective when properly managed, requiring routine viral load monitoring to assess successful viral suppression. Efforts to expand access by decentralizing HIV nucleic acid testing in low- and middle-income countries has been hampered by the cost and complexity of current tests. Sample preparation of blood samples has traditionally relied on cumbersome RNA extraction methods, and it continues to be a key bottleneck for developing low-cost POC nucleic acid tests. We present a microfluidic paper-based analytical device (µPAD) for extracting RNA and detecting HIV in serum, leveraging low-cost materials, simple buffers, and an electric field. We detect HIV virions and MS2 bacteriophage internal control in human serum using a novel lysis and RNase inactivation method, paper-based isotachophoresis (ITP) for RNA extraction, and duplexed reverse transcription recombinase polymerase amplification (RT-RPA) for nucleic acid amplification. We design a specialized ITP system to extract and concentrate RNA, while excluding harsh reagents used for lysis and RNase inactivation. We found the ITP µPAD can extract and purify 5,000 HIV RNA copies per mL of serum. We then demonstrate detection of HIV virions and MS2 bacteriophage in human serum within 45-minutes.</p>

HIV Detection from Human Serum with Paper-Based Isotachophoretic RNA Extraction and Reverse Transcription Recombinase Polymerase Amplification

2021 ◽  
Author(s):  
Andrew Bender ◽  
Benjamin Sullivan ◽  
Jane Zhang ◽  
David Juergens ◽  
Lorraine Lillis ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Human Serum ◽  
Internal Control ◽  
Reverse Transcription ◽  
Low Cost ◽  
Rna Extraction ◽  
Recombinase Polymerase Amplification ◽  
Nucleic Acid Amplification ◽  
People Living With Hiv ◽  
Ms2 Bacteriophage

<p>The number of people living with HIV continues to increase with the current total near 38 million, of which about 26 million are receiving antiretroviral therapy. These treatment regimens are highly effective when properly managed, requiring routine viral load monitoring to assess successful viral suppression. Efforts to expand access by decentralizing HIV nucleic acid testing in low- and middle-income countries has been hampered by the cost and complexity of current tests. Sample preparation of blood samples has traditionally relied on cumbersome RNA extraction methods, and it continues to be a key bottleneck for developing low-cost POC nucleic acid tests. We present a microfluidic paper-based analytical device (µPAD) for extracting RNA and detecting HIV in serum, leveraging low-cost materials, simple buffers, and an electric field. We detect HIV virions and MS2 bacteriophage internal control in human serum using a novel lysis and RNase inactivation method, paper-based isotachophoresis (ITP) for RNA extraction, and duplexed reverse transcription recombinase polymerase amplification (RT-RPA) for nucleic acid amplification. We design a specialized ITP system to extract and concentrate RNA, while excluding harsh reagents used for lysis and RNase inactivation. We found the ITP µPAD can extract and purify 5,000 HIV RNA copies per mL of serum. We then demonstrate detection of HIV virions and MS2 bacteriophage in human serum within 45-minutes.</p>

scholarly journals Development of an Automated, Non-Enzymatic Nucleic Acid Amplification Test

Micromachines ◽  
2021 ◽  
Vol 12 (10) ◽  
pp. 1204
Author(s):  
Zackary A. Zimmers ◽  
Alexander D. Boyd ◽  
Hannah E. Stepp ◽  
Nicholas M. Adams ◽  
Frederick R. Haselton
Keyword(s):  
Nucleic Acid ◽  
Internal Control ◽  
Magnetic Beads ◽  
Point Of Care ◽  
Dna Amplification ◽  
Magnetic Bead ◽  
Nucleic Acid Amplification ◽  
Diagnostic Strategies ◽  
Low Resource ◽  
Target Dna

Among nucleic acid diagnostic strategies, non-enzymatic tests are the most promising for application at the point of care in low-resource settings. They remain relatively under-utilized, however, due to inadequate sensitivity. Inspired by a recent demonstration of a highly-sensitive dumbbell DNA amplification strategy, we developed an automated, self-contained assay for detection of target DNA. In this new diagnostic platform, called the automated Pi-powered looping oligonucleotide transporter, magnetic beads capture the target DNA and are then loaded into a microfluidic reaction cassette along with the other reaction solutions. A stepper motor controls the motion of the cassette relative to an external magnetic field, which moves the magnetic beads through the reaction solutions automatically. Real-time fluorescence is used to measure the accumulation of dumbbells on the magnetic bead surface. Left-handed DNA dumbbells produce a distinct signal which reflects the level of non-specific amplification, acting as an internal control. The autoPiLOT assay detected as little as 5 fM target DNA, and was also successfully applied to the detection of S. mansoni DNA. The autoPiLOT design is a novel step forward in the development of a sensitive, user-friendly, low-resource, non-enzymatic diagnostic test.

A national quality assurance survey of Neisseria gonorrhoeae testing

2014 ◽  
Vol 63 (1) ◽  
pp. 45-49 ◽  
Author(s):  
Ella Trembizki ◽  
Monica Lahra ◽  
Kerrie Stevens ◽  
Kevin Freeman ◽  
Tiffany Hogan ◽  
...  
Keyword(s):  
Quality Assurance ◽  
Nucleic Acid ◽  
Neisseria Gonorrhoeae ◽  
Nucleic Acid Amplification ◽  
Reference Standard ◽  
Gene Sequences ◽  
Surveillance Programme ◽  
National Quality ◽  
Rrna Sequencing ◽  
Pcr Assays

The aims of this study were to (1) conduct a national survey of Neisseria gonorrhoeae identification by National Neisseria Network (NNN) reference laboratories contributing data to the Australian Gonococcal Surveillance Programme and (2) determine the prevalence in Australia of strains of N. gonorrhoeae lacking gene sequences commonly targeted by in-house PCR assays for confirmation of gonococcal nucleic acid amplification tests. Gonococcal clinical isolates referred to NNN laboratories for the first half of 2012 were screened using in-house real-time PCR assays targeting multicopy opa, porA pseudogene and cppB genes. There were 2455 clinical gonococcal isolates received in the study period; 98.6 % (2420/2455) of isolates harboured all three gene targets, 0.12 % (3/2455) were porA-negative, 0.04 % (1/2455) opa-negative and 1.14 % (28/2455) cppB-negative by PCR. Notably, no isolates were simultaneously negative for two targets. However, three isolates failed to be amplified by all three PCR methods, one isolate of which was shown to be a commensal Neisseria strain by 16S rRNA sequencing. Using PCR as the reference standard the results showed that (1) identification of N. gonorrhoeae isolates by NNN laboratories was highly specific (99.96 %) and (2) strains of N. gonorrhoeae lacking gene sequences commonly targeted by in-house PCR assays are present but not widespread throughout Australia at this point in time.

scholarly journals Diagnostic Use of PCR in Carbapenamase-producing Enterobacteriaceae (CPE): An Improved Method to Overcome the presence of inhibitors for DNA Extraction from Blood Cultures

2018 ◽  
Author(s):  
Zeti Norfidiyati Salmuna ◽  
Murnihayati Hassan
Keyword(s):  
Nucleic Acid ◽  
Dna Extraction ◽  
Internal Control ◽  
Previous Method ◽  
Nucleic Acid Amplification ◽  
Culture Bottle ◽  
Predominant Genotype ◽  
Specificity And Sensitivity ◽  
Nucleic Acid Amplification Technology ◽  
Amplification Technique

AbstractBackgroundCarpanenamase-producingEnterobacteriaceae(CPE) has emerged as a threat to hospitalized patients. Phenotypic test such as Modified hodge test was less sensitive and specific especially to detectblaNDM-1 which is the most predominant genotype in this region. Nucleic acid amplification technology offers improved specificity and sensitivity. However, there is limitation in this method which is failed amplification due to the presence of inhibitors. In this study, we try to use previous method described by Villumseen et al with some modification using another DNA extraction kit.Methodology/Principle findingsThree confirmed isolates ofblaNDM-1 carbapenamase-producingKlebsiella pneumoniaewas spiked with 10 mls sterile whole blood in an aerobic Bactec Plus until the growth was detected. The blood specimen was taken and was subjected to DNA extraction method using two commercialized kits followed with multiplex PCR. All the three isolates revealedblaNDM-1 genotypes. Internal control was amplified in all three isolates.Conclusion/significanceThis study proved that we can get a specific and early diagnosis of CPE by using nucleic acid amplification technique directly from blood culture bottle and eliminate the effect of inhibitors.

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