iCubate

PCR, qPCR and mPCR

No one can imagine biotech without PCR. There are many variations of the basic PCR technology, just visit the Wikipedia link, to find more than 20 different PCR based methods.

qPCR or real time PCR, is one of the most popular technologies today. It is popular because it has several desired features. It is sensitive, quantitative, and it integrates amplification and detection. However, it is very difficult to develop a multiplex assay for qPCR.

We have been focused on developing mPCR (multiplex PCR) technologies for the past 10 years. We first invented the patented tem-PCR technology (for target enriched multiplex PCR technology). In tem-PCR (see figure below), nested primers were designed for each targets needing to be co-amplified (Fo, Fi, Ro and Ri) in the multiplex assay, the inside primers for all the targets share the same tag sequence which will bind to a pair of “Superprimers” (FS and RS). All these nested primers and the superprimers are in the same reaction system together, though the nested gene specific primers are used at extremely low concentration. Only the superprimers are at high concentration for exponential amplification of the targets. The tem-PCR technology worked because it solved the primer (loci) incompatibility problem. We used the tem-PCR technology successfully to develop several multiplex PCR assays for infectious diseases diagnoses (references 1-12).

In 2009, we developed the next generation mPCR technology, the arm-PCR (amplicon rescued multiplex PCR, patent pending). The arm-PCR technology was invented to work with iCubate integrated system. For arm-PCR (see figure below), we are using a high concentration of the nested primers in the initial PCR round. Then, we will rescue the amplicon and perform a second round of PCR by adding fresh communal primers (recognizing the shared tag sequence already introduced during the first round of amplification) and enzymes. The two rounds of reactions are set up automatically in the iCubate cassette, so that we do not need to worry about open the reaction tube and contaminate the laboratory environment with a high concentration of amplicons.

One major difference between qPCR and mPCR is that the former detects amplification at real time, where mPCR performs end point analysis. In order to perform quantitative measurement, the Ct value (which is the number of PCR cycles that elapse before the threshold is reached) is often used (see chart below). By comparing the results of samples of unknown concentration with a series of standards, the amount of template DNA in an “unknown” reaction can be accurately determined.

Looking at the figure above, you will conclude that end point analysis could never be quantitative, since it does not matter what starting concentration you may have. After the reaction reached the plateau (after 40 cycles), there is almost no difference in signal levels.

But in mPCR, since the exponential phase of the amplification is carried out by one pair of primers for all the potential targets, the plateau is established by the contributions from all the targets in the reaction system. Therefore, end point analysis could become semi-quantitative. This feature was used in our recent study of using arm-PCR to amplify immune repertoire for high throughput sequencing (Wang et al., 2010).

In many cases, such as infectious disease differential diagnosis, mPCR is not used to amplify all the intended targets at once, but only anticipating one or two will be amplified from many possibilities. In that case, the assay is a qualitative one with a cutoff value.

References:

  1. Jian Han,  David C. Swan, Sharon J. Smith, Shanjuan H. Lum, Susan E. Sefers, Elizabeth R. Unger, and Yiwei Tang. (2006) Simultaneous Amplification and Identification of 25 Human Papillomavirus Types with Templex Technology. Journal of Clinical Microbiology. 44(11). 4157-4162.
  2. Han J. Molecular differential diagnoses of infectious diseases: is the future now? In: Stratton C. and Tang YW. Eds. Advanced Technologies in Diagnostic Microbiology. New York. NY. Springer Publishing Company. (2006)
  3. John Brunstein, and Eva Thomas. (2006) Direct Screening of Clinical Specimens for Multiple Respiratory Pathogens Using the Genaco Respiratory Panels 1 and 2. Diagn Mol Pathol. 15(3). 169-173.
  4. Haijing Li, Melinda A. McCormac, R. Wray Estes, Susan E. Sefers, Ryan K. Dare, James D. Chapell, Dean D. Erdman, Peter F. Wright, and Yi-Wei Tang. (2007) Simultaneous Detection and High-Throughput Identification of a panel of RNA viruses Causing Respiratory Tract Infections. Journal of Clinical Microbiology, July. 45(7) 2105-2109.
  5. Shumei Zou, Jian Han, Leying Wen, Yan Liu, Kassi Cronin, Shanjuan H. Lum, Lu Gao, Jie Dong, Ye Zhang, Yuanji Guo and Yuelong Shu. (2007) Human Influenza A Virus (H5N1) Detection by a Novel Multiplex PCR Typing Method. Journal of Clinical Microbiology. 45(6). 1889-1892.
  6. Yi-Wei Tang, Abdullah Killic, Qunying Yang, Sigrid K. McAllister, Haijing Li, RebeccaS. Miller, Melinda McCormac, Karen D. Tracy, Charles W. Stratton, Jian Han, and Brandi Limbago. (2007) StaphPlex System for Rapid and Simultaneous Identification of Antibiotic Resistance Determinants and Panton-Valentine Leukocidin Detection of Staphylococci from Positive Blood Cultures. Journal of Clinical Microbiology. 45(6). 1867-1873.
  7. Kenneth L. Muldrew., Safedin H. Beqaj, Jian Han, Shanjuan H. Lum, Vicki Clinard, Stephen J. Schultenover, and Yiwei Tang. (2007)  Evaluation of a Digene-Recommended Algorithm for Human Papillomavirus Low-Positive Results Present in a “Retest Zone”. American Journal of Clinical Pathology. 127: 97-102.
  8. Medea Gegia, Nino Mdivani, Rodrigo E. Mendes, Haijing Li, Maka Akhalaia, Archil Salakaia, Jian Han, George Khechinashvili, and Yi-Wei Tang. Prevalence of and Molecular Basis for Tuberculosis Drug Resistance in the Republic of Georgia: Validation of a QIAplex System for Detection of Drug Resistance-Related Mutations. Antimicrobial Agents and Chemotherapy, Feb. 2008, p 725-729
  9. John Brunstein, Christy L. Cline, Steven McKinney, and Eva Thomas. Evidence from Multiplex Molecular Assay for Complex Multi-pathogen Interactions in Acute Respiratory Infections. Journal of Clinical Microbiology, Jan. 2008, p. 97-102.
  10. Robert Benson, Maria L. Tondella, Julu Bhatnagar, Maria da Gloria S. Carvalho, Jacquelyn S. Sampson, Deborah F. Talkington, Anne Whitney, Elizabeth mothershed, Lesley McGee, George Carlone, Vondguraus McClee, Jeannette Guarner, Sherif Zaki, Surang Dejsiri, Kassi Cronin, Jian Han and Barry Fields. Development and Evaluation of a Novel Multiplex PCR Technology for Molecular Differential Detection of Bacterial Respiratory Disease Pathogens. Journal of Clinical Microbiology, June 2008, p.2074-2077
  11. Edward H. Eiland III, Nicholas Beyda, Jian Han, William Lindgren, Randy Ward, Thomas MacAndrew English, Ali Hassoun, and Kathi Hathcock. The Utility of Rapid Micorbiological and Molecular Techniques in Optimizing Antimicrobial Therapy. SRX Pharmacology 2010, Article ID 395215.
  12. Chunlin Wang, Catherine M. Sanders, Qunying Yang, Harry W. Schroeder, Jr., Elijah Wang, Farbod Babrzadeh, Baback Gharizadeh, Richard M. Myers, James R. Hudson, Jr., Ronald W. Davis, and Jian Han. High throughput sequencing reveals a complex pattern of dynamic interrelationships among human T cell subsets. PNAS Jan 26, 2010 vol. 107(4) 1518-1523.
  13. Kassi Koon, Catherine M. Sanders, Jessica Green, Leslie Molone, Holly White, Delineliz Zayas, Rebecca Miller, Stanley Lu and Jian Han. Co-detection of Pandemic (H1N1) 2009 Virus and Other Respiratory Pathogens. Emerging Infectious Diseases.  2011 vol. 16 (12) 1976-1978
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