What is pcr AND what is qrt-pcr?
Polymerase Chain Reaction (PCR)
Polymerase Chain Reaction (PCR) is a molecular biology technique that amplifies a specific DNA segment, resulting in the production of millions of copies of that DNA sequence.
The technique was invented by Kary Mullis in 1983 and has since revolutionized the fields of genetics, forensic science, and medical diagnosis.
Quantitative, real-time polymerase chain reaction (qRT-PCR) is a variant of PCR that allows for the quantification of the initial amount of DNA template in a sample.
qRT-PCR involves the use of a fluorescent dye or a fluorescently labeled probe that binds to the amplified DNA product as it is being produced in real-time.
As more DNA is amplified, the fluorescent signal increases proportionally, allowing for the quantification of the starting amount of DNA.
The PCR technique is highly sensitive and specific, making it a valuable tool for the detection and quantification of pathogens, genetic mutations, and gene expression levels.
To perform qRT-PCR, RNA is first isolated from the sample of interest, and then converted into complementary DNA (cDNA) using reverse transcriptase enzyme.
The cDNA is then used as the template for PCR amplification. During amplification, a fluorescently labeled probe or a fluorescent dye is incorporated into the newly synthesized DNA strands, and the fluorescence signal is measured in real-time using a specialized instrument called a real-time PCR instrument.
The resulting data is analyzed using software that generates a standard curve from a series of dilutions of a known DNA template. This standard curve is then used to determine the starting amount of DNA in the original sample.
qRT-PCR has numerous applications in both basic and clinical research.
It can be used to quantify gene expression levels, identify genetic mutations, and detect the presence of infectious agents.
The technique has been particularly useful in the field of medical diagnostics, where it is used to detect and monitor viral infections such as HIV, hepatitis, and COVID-19.
In conclusion, qRT-PCR is a powerful analytical method that allows for the precise quantification of DNA in a sample. Its sensitivity, specificity, and versatility make it an essential tool in molecular biology research and clinical diagnostics.
Specificity and Sensitivity of PCR Testing
Culture Media Testing vs PCR Testing
Culture growth media and PCR testing are both accurate methods for identifying the presence of bacteria, viruses, and other microorganisms. However, culture growth media is often a more sensitive and reliable method for detecting microorganisms, as it can detect even small amounts of bacteria or viruses. PCR testing, on the other hand, is more specific and can detect specific sequences of DNA or RNA. PCR testing is also faster than culture growth media, and can be used to detect the presence of microorganisms in a shorter period of time.
Number of days to detect Listeria with Culture Growth media
It typically takes between 24 and 48 hours to detect Listeria in a culture growth media. However, it can take longer depending on the type of culture media used, the number of organisms present, the growth conditions, and other factors. Additionally, some types of culture media may require enrichment before the organisms can be detected. In general, it is best to follow the instructions provided with the culture media for the best results.
Is a direct-to-PCR test accurate?
Yes, a no pre-enrichment PCR test (direct-to-PCR Test) can be accurate. PCR testing is a highly sensitive method of detecting the presence of DNA or RNA, and can detect even small amounts of the target sequence. PCR testing does not require pre-enrichment, meaning that it can detect the presence of a target organism without the need for growth or incubation. However, it is important to follow the instructions provided with the PCR test to ensure accurate results.
Is a pre-enrichment PCR test more accurate?
Yes, in general, a pre-enrichment PCR test can be more accurate than a direct-to-PCR test for detecting human pathogens. Pre-enrichment PCR tests are designed to detect even small amounts of the target sequence, as they can detect the presence of organisms after they have been incubated for a period of time. This can lead to more accurate results, as the organisms have had more time to grow and multiply, making them easier to detect. Direct-to-PCR tests, on the other hand, do not require pre-enrichment, and may not be as sensitive as pre-enrichment tests, as they may not be able to detect small amounts of the target sequence.
Is the Cepheid SmartCycler System reliable?
Yes, the Cepheid SmartCycler platform is considered to be a viable DNA detection system. It is a PCR-based system that can be used to detect and quantify DNA sequences in a wide range of applications, including diagnostics and research. The SmartCycler platform has been designed to provide accurate and reliable results and can detect even small amounts of DNA, making it a viable option for detecting and quantifying DNA. Additionally, the SmartCycler can be used for a variety of other applications, such as genotyping, gene expression analysis, and mutation detection.
Can non-degreed persons be trained to run PCR tests?
Yes, non-degreed technicians can be trained to perform PCR testing. They will need to be trained in the appropriate laboratory techniques and safety protocols. Additionally, they should receive adequate training in the specific PCR tests that they are performing, including the proper use of the equipment, the calculation of sample concentrations, and the interpretation of test results. Once they have been adequately trained and certified, technicians should be able to perform the test correctly and accurately.
How accurate is a qRT-PCR test result report?
The accuracy of a Cycle Threshold (Ct) PCR test result report depends on a number of factors, including the accuracy of the laboratory equipment, the quality of the sample, and the expertise of the technician performing the test. Generally, PCR tests are considered to be highly accurate if the appropriate protocols and procedures are followed. If the Ct value of the PCR test result is within the expected range for the sample, it is likely to be accurate. However, it is important to note that PCR tests may not be able to detect small amounts of the target sequence, so it is important to consult the instructions for the specific test to determine the expected range of Ct values.
Cepheid Smartcycler on YouTube
YouTube videos & Cepheid Smart Cycler:
"What is Cepheid's GeneXpert System?", https://www.youtube.com/watch?v=k-m4Z4Q2A5A
"What is the Smart Cycler System?", https://www.youtube.com/watch?v=cJU6jCjl6lM
"Cepheid Smart Cycler: Making Diagnostics Easier", https://www.youtube.com/watch?v=Uv7bUo9XKjY
Cepheid Technology & US Food & Drug Administration
FDA's Mobile Laboratories
Mobile laboratories help FDA save time and quickly test food and drugs for contaminants.
April 8, 2009; Nogales, AZ – Working on-site with samples collected by FDA consumer safety officers from produce shipments crossing the border, an FDA mobile lab analyst tests for Salmonella and E. coli.
Mobile laboratories help FDA quickly test food and drugs for contaminants. When not deployed, the mobile labs are stationed at FDA's Jefferson, Arkansas Regional Laboratory. Learn more at www.fda.gov
https://www.flickr.com/photos/fdaphotos/albums/72157625637329385
LLNL/CEPHEID SMARTCYCLER DEVELOPMENT
Technology originally developed by Lawrence Livermore National Laboratories (LLNL)
Confirmatory diagnostic methods utilizing polymerase chain reaction (PCR) methodologies in tandem with innovative, state-of-the-art biosensor technologies.
Blakely, William & Brooks, Antone & Lofts, Richard & Schans, Govert & Voisin, Philippe. (2002). Overview of Low-Level Radiation Exposure Assessment: Biodosimetry. Military medicine. 167. 20-4. 10.1093/milmed/167.suppl_1.20.
Cepheid's battery-operated. notebook Smart Cycler, partially funded by U.S. Army Medical Research Institute of Infectious Diseases, which performs nucleic extraction and PCR assay in an integrated handheld device.. A schematic of a handheld biosensor, which is based on an optical waveguide design that detects changes in the relative refractive index of multiple biomatrix antibody/antigen complexes. The Sunnyvale company Cepheid, for example, used Lab technology to develop the Smart Cycler DNA testing system, which has been beneficial to the nation's anti-terrorism efforts. The system was part of the Livermore/LANL jointly developed Biological Aerosol Sentry and Information System (BASIS).
Michael L. Perdue "Molecular Diagnostics in an Insecure World," Avian Diseases 47(s3), 1063-1068, (1 September 2003). https://doi.org/10.1637/0005-2086-47.s3.1063
The Joint Biological Agent Identification and Diagnostic System (JBAIDS) Block I began fielding in 2006. JBAIDS is a fully integrated in vitro diagnostic system composed of the JBAIDS instrument with laptop computer, software, freeze-dried reagent assays and sample preparation protocols for isolating target DNA from whole blood, blood culture, or direct culture. The JBAIDS instrument, using Polymerase Chain Reaction (PCR) technology, is a portable thermocycler and real-time fluorimeter
https://irp.fas.org/threat/cbdp2007.pdf
Journal for the Association for Laboratory Automation. Volume 3, No.6, December 1998
https://journals.sagepub.com/doi/pdf/10.1177/221106829800300604
SmartCycler on-board the International Space Station
Cepheid SmartCycler on the ISS
NASA astronaut and Expedition 47 flight engineer Jeff Williams works with the Wet Lab RNA SmartCycler on-board the International Space Station. Wetlab RNA SmartCycler is a research platform for conducting real-time quantitative gene expression analysis aboard the ISS. The system enables spaceflight genomic studies involving a wide variety of biospecimen types in the unique microgravity environment of space.
Gene Expression Analysis in a Microgravity Environment
https://ntrs.nasa.gov/citations/20150018249
https://www.flickr.com/photos/nasa2explore/25941659244/in/photolist-FwnMuY/
Toward biotechnology in space: High-throughput instruments for in situ biological research beyond Earth
Validation of the International Space Station Smart Sample Concentrator for Microbial Monitoring of Low Biomass Water Samples
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7566613/
WetLab-2 employs a standard method of measuring gene expression called Quantitative Polymerase Chain Reaction, or qPCR, which involves extracting certain types of ribonucleic acid (RNA) molecules from biological samples and then measuring the amounts extracted.
https://www.nasa.gov/mission_pages/station/research/news/wet_lab2
NASA astronaut and Expedition 47 Flight Engineer Jeff Williams works with the Wet Lab RNA SmartCycler on-board the International Space Station. Wetlab RNA SmartCycler is a research platform for conducting real-time quantitative gene expression analysis aboard the ISS. The system enables spaceflight genomic studies involving a wide variety of biospecimen types in the unique microgravity environment of space.
https://spaceref.com/space-stations/nasa-international-space-station-on-orbit-status-19-april-2016/
SmartCycler-based RT PCR Leishmania assay
RT PCR Leishmania assay
The Leishmania Diagnostics Laboratory at Walter Reed Army Institute of Research (WRAIR) of the US Army has utilized the College of American Pathologists-certified SmartCycler-based RT PCR Leishmania assay for diagnosis of cutaneous leishmaniasis cases in theater, and efforts are ongoing to obtain U.S. FDA licensure for this product (Wortmann et al. 2005). A rapid diagnostic assay to detect scrub typhus in humans is in development.
https://apps.dtic.mil/sti/pdfs/ADA502972.pdf
Wortmann, G., L. Hochberg, H.-H. Houng, C. Sweeney, M. Zapor, N. Aronson, P. Weina, and C.F. Ockenhouse. 2005. Rapid identification of Leishmania complexes by a real-time PCR assay. Am. J. Trop. Med. Hyg. 73: 999- 1004.
US FDA BAM Chapter 4A: Diarrheagenic Escherichia coli
Screening method for E. coli STEC and Serotype O157:H7 from Foods
Food samples may be screened for O157:H7 using ....the SmartCycler II.
Escherichia coli is one of the predominant species of facultative anaerobes in the human gut and usually harmless to the host; however, a group of pathogenic E. coli has emerged that causes diarrheal disease in humans. Referred to as Diarrheagenic E. coli or commonly as pathogenic E. coli, these groups are classified based on their unique virulence factors and can only be identified by these traits. Hence, analysis for pathogenic E. coli often requires that the isolates be first identified as E. coli before testing for virulence markers [Enterohemorrhagic Escherichia coli (EHEC)].
The real-time PCR assay configured for the SmartCycler II platform, is specific for the stx1 and stx2 genes and the +93 single nucleotide polymorphism in the uidA gene that encode for the β-D-glucuronidase (GUD) enzyme (11). The +93 SNP is highly conserved in O157:H7 and O157:H- strains that produce Stx and is an accurate identification marker for O157:H7 strains. The stx1 and stx2 markers on the real-time PCR assays also enabled the detection of other STEC strains, some of which are known human pathogens
https://www.fda.gov/food/laboratory-methods-food/bam-chapter-4a-diarrheagenic-escherichia-coli
The US Food and Drug Administration (FDA) uses the Cepheid SmartCycler platform for the analysis of samples in their laboratories. The SmartCycler is used to perform PCR tests on DNA samples, including tests for the detection of infectious diseases. The FDA has also approved the use of the SmartCycler platform for the detection of certain foodborne pathogens, including Salmonella and Listeria. Additionally, the SmartCycler is used to perform tests for the detection of genetic mutations associated with certain diseases, such as cystic fibrosis and sickle cell anemia.
The US Food and Drug Administration (FDA) uses the Cepheid SmartCycler platform to perform PCR tests on DNA samples, including tests for the detection of infectious diseases, the detection of foodborne pathogens such as Salmonella and Listeria, and the detection of genetic mutations associated with certain diseases, such as cystic fibrosis and sickle cell anemia. Additionally, the FDA also uses the SmartCycler platform to perform tests for the assessment of fibrosis resulting from HCV infection [1], the detection of DNA from Bacteroidales bacteria in ambient water matrices [2], and the detection of viral nucleic acid in samples. Hepatitis C Diagnostics Technology Landscapehttps://unitaid.org/assets/UNITAID-HCV_Diagnostic_Landscape-1st_edition-1.pdf Method B: Bacteroidales in Water by TaqMan Quantitative ...https://www.epa.gov/sites/default/files/2015-08/documents/method_b_2010.pdf
direct-to-pcr, quantitative, real-time pcr
DTPCR As Accurate As Pre-Enrichment
Direct-to-pcr, quantitative, real-time polymerase chain reaction is as accurate as pre-enrichment PCR.
Studies evaluated the accuracy and reliability of direct-to-PCR and pre-enrichment PCR methods for detecting various bacterial pathogens in different sample types.
The direct-to-PCR method is as accurate as pre-enrichment PCR for detecting pathogens.
The accuracy and reliability of direct-to-PCR and pre-enrichment PCR depend on the specific pathogen, sample type, and specific PCR assay employed to detect for either the presence/absence or quantitation of the target(s).
USDA and the Cepheid smartcycler
The USDA uses the Cepheid SmartCycler
The US Department of Agriculture (USDA) uses the Cepheid SmartCycler platform for the analysis of samples in their laboratories. The SmartCycler is used to perform PCR tests on DNA samples, including tests for the detection of foodborne pathogens, such as Salmonella, E. coli, and Listeria. Additionally, the USDA also uses the SmartCycler platform to perform tests for the detection of genetic mutations associated with certain diseases, such as cystic fibrosis and sickle cell anemia.
Which tests does the USDA run on the SmartCycler?
The US Department of Agriculture (USDA) uses the Cepheid SmartCycler platform to perform PCR tests on DNA samples, including tests for the detection of foodborne pathogens, such as Salmonella, E. coli, and Listeria. Additionally, the USDA also uses the SmartCycler platform to perform tests for the detection of genetic mutations associated with certain diseases, such as cystic fibrosis and sickle cell anemia, as well as tests for the detection of viral nucleic acid in samples and the assessment of fibrosis resulting from HCV infection.
The USDA uses the unit in the field
The Cepheid SmartCycler platform can be used in field operations. The device is designed to be portable and can be used in a variety of settings, including in the laboratory, in healthcare settings, and in the field. The device is easy to use and requires minimal training, making it ideal for use in the field. Additionally, the SmartCycler can be used to perform a variety of tests, including PCR tests for the detection of infectious diseases and genetic mutations associated with certain diseases, as well as tests for the detection of foodborne pathogens and viral nucleic acid.
Historical PCR & AME’s qRT-PCR & DTPCR Systems
Evolution to Current PCR Systems
Originally polymerase chain reaction (PCR) experiments required four (4) different rooms for sample pre-enrichment preparation, sample extraction, reagent mixing, amplification of the sample, and test results analysis, however, with the advent of real-time pcr pre-mixed kits and direct-to-pcr testing the engagement of rt-pcr testing has been greatly simplified.
Polymerase chain reaction (PCR) is a molecular biology technique used for the amplification of DNA sequences. Originally, PCR experiments required four different rooms for sample pre-enrichment preparation, sample extraction, reagent mixing, amplification of the sample, and test results analysis.
With the advent of real-time PCR pre-mixed kits and direct-to-PCR testing, the engagement of PCR testing has been greatly simplified. In this article, we will discuss the evolution of the PCR testing method and how it has changed over time.
PCR was first introduced in 1983 by Kary Mullis, and it quickly became an indispensable tool in molecular biology. The original method involved a series of temperature cycles to denature DNA, anneal primers, and extend the DNA using a DNA polymerase enzyme. The PCR process was time-consuming and required expertise in DNA extraction, primer design, and PCR optimization.
Over time, PCR technology has evolved, and various modifications have been made to the original method to improve its efficiency and sensitivity. One of the most significant improvements has been the development of real-time PCR, which allows the detection of PCR amplification in real-time, as it happens. Real-time PCR has several advantages over traditional PCR, including increased speed, sensitivity, and specificity.
Real-time PCR has been made possible by the development of pre-mixed kits that contain all the necessary reagents for PCR amplification. These kits are designed to simplify the PCR process by reducing the need for sample pre-enrichment and DNA extraction. This has made PCR testing more accessible, faster, and more reliable.
The direct-to-PCR approach, which allows for the amplification of DNA directly from crude samples, without the need for DNA extraction. This approach has significantly reduced the time and cost required for PCR testing, making it more accessible for clinical and diagnostic applications.
PCR technology has also evolved to allow for multiplex PCR, which can detect multiple DNA targets simultaneously. This has made PCR testing more efficient, especially in pathogen detection.
PCR technology has also been adapted for use in point-of-care (POC) testing, where it can be used to diagnose infectious diseases and monitor treatment outcomes in real-time. POC testing has the potential to improve patient outcomes by allowing for early diagnosis and timely treatment.
In conclusion, the evolution of PCR technology has simplified the PCR testing process, making it faster, more efficient, and more accessible for clinical and diagnostic applications. The development of real-time PCR, pre-mixed kits, direct-to-PCR, multiplex PCR, and POC testing has revolutionized PCR technology, and it continues to be a valuable tool in molecular biology and medical diagnostics, especially during the recent COVID-19 pandemic.
Identifying bacterial pathogen's dna sequence via ngs
AME DNA Identification Capability
AME's capability's to generate Whole Genome Sequencing (WGS) and Next Generation Sequencing (NGS) data from collected samples which can then be employed to confirm beyond any doubt that bacterial pathogens were traced back to the source in finished products, raw products, and production facilities is unique and confirming beyond any doubt.
Absolute Sub-Speciation of Samples
Next Generation Sequencing (NGS) determines the sequence of a given nucleic acid strand, such as DNA of a sample. This technology can identify with specificity whether or not the specific DNA originated in a food sample either to determine the source for culpability or exoneration by a food producer.
Scientific Data to Support Claims
Claiming parties must prove the causation of a harmful bacterial pathogens as originating from a food producer before being awarded financial compensation.
Lack of a DNA link from physical evidence will successfully defend innocent food producers. NGS data can be key to defending such unsupportable claims.
For further information:
Andy Moreno, PhD
Bacterial Surveillance Systems Engineer
AME Certified PCR Laboratories
650-445-4115