Polymerase chain reaction(PCR) was born in the year of 1985, which amplifies DNA through denaturation, annealing and extension, simulating in vivo DNA replication progress. PCR has radically altered molecular biology; via this technique, a trace amount of DNA can be amplified by tens of orders of magnitude, thus to produce adequate products for analysis.
Several variations of PCR have been invented: (1) Nested PCR incorporates two pairs of primers flanking the same target sequence, which increases the sensitivity, i.e., the limit of detection (LOD); (2) RT-PCR monitors the fluorescence during the amplification, predicting the initial template’s abundance; (3) Multiplexed PCR amplifies multiple DNA fragments simultaneously. All these methods need a thermal cycler; Nested PCR and Multiplexed PCR need more than two pairs of primers, which may lead to the generation of primer-dimers; RT-PCT needs expensive CCD to capture signals in real time.
In order to overcome the disadvantages of the conventional PCR methods, effective, sensitive, and instrument-free amplification methods have been developed in the past decades: such as nucleic acid sequence-based amplification (NASBA), transcription mediated amplification (TMA), signal mediated amplification of RNA technology (SMART), strand displacement amplification (SDA), rolling circle amplification (RCA), loop-mediated isothermal amplification of (LAMP), multiple displacement amplification (MDA), and helicase dependent amplification (HDA). All of them amplify nucleic acid sequence rapidly. To further ameliorate our detecting system, we could bring efficient isothermal PCR in the project.
1. Multiple displacement amplification (MDA)
MDA uses phi29 DNA Polymerase and random primers to amplify template DNA isothermally at 30℃, which produces 10,000 fold amplification after overnight incubation. This reaction utilizes the strand displacement activity and great processivity of phi29 DNA Polymerase, the former makes it possible to amplify DNA without denaturing, the latter allows it to synthesize DNA strands as long as 70,000bp in length without dissociation (Figure 1).
MDA generates DNA products robustly in an average length of over 10Kb even up to 100Kb (Figure 2); this robustness to replicate through a complex sequence and a wide coverage makes it to evenly amplify the target, regardless of DNA primary or secondary structure. In the end of amplification, there is about 20–30 ug products from as few as 1–10 copies of human genomic DNA. In previous studies it was suggested that pyrophosphatase be added to the reaction to eliminate the inhibitory accumulation of pyrophosphate for a higher yield. For phi29 DNA polymerase has 3’ to 5’ exonuclease activity, exonuclease-resistant primers should be exploited during the amplification, such as thiophosphate-modified random hexamer (5’-NpNpNpNpsNpsN-3’); this further augments the amount of DNA products.
MDA eliminates elaborate template preparation procedure; amplification could be carried out directly from the crude cells, blood lysates, marrow aspirates, and buccal swabs, even stored DNA such as paraffin-embedded tissues. This also eliminates manual errors and is beneficial for district where there lacks technical guidance. The incubation time is from 2h to 16h, qualifying for rapid detection of pathogen. As we have shown, the required concentration of DNA samples for our PC Reporter is relatively low, so we could further reduce the incubation time.
Figure 1. In MDA system, when random primers anneal to the template DNA, phi29 DNA polymerase extends the strand, displaces downstream strands via its strand displacement activity and produces branched structures through its great processvity. This eventually produces hyperbranched products, which can be 10Kb, even exceeds 100Kb in length.
Figure 2.MDA to amplify two different targets. A significant fraction of DNA products is too large to leave the sample well during electrophoresis. M for Marker, 1 for product of pSB1C3, 2 for pSB1C3, 3 for product of pET21a, 4 for pET21a, 5 for H2O negative control.
References
1 Dean, F. B., Hosono, S., Fang, L., Wu, X., Faruqi, A. F., Bray-Ward, P., ... & Lasken, R. S. (2002). Comprehensive human genome amplification using multiple displacement amplification.Proceedings of the National Academy of Sciences,99(8), 5261-5266.
2 Esteban, J. A., Salas, M., & Blanco, L. (1993). Fidelity of phi 29 DNA polymerase. Comparison between protein-primed initiation and DNA polymerization. Journal of Biological Chemistry,268(4), 2719-2726.
3 Dean, F. B., Nelson, J. R., Giesler, T. L., & Lasken, R. S. (2001). Rapid amplification of plasmid and phage DNA using phi29 DNA polymerase and multiply-primed rolling circle amplification. Genome research, 11(6), 1095-1099.
4 Luthra, R., & Medeiros, L. J. (2004). Isothermal multiple displacement amplification: a highly reliable approach for generating unlimited high molecular weight genomic DNA from clinical specimens. The Journal of Molecular Diagnostics,6(3), 236-242.
5 Lasken, R. S., & Egholm, M. (2003). Whole genome amplification: abundant supplies of DNA from precious samples or clinical specimens.Trends in biotechnology, 21(12), 531-535.
6 Aviel-Ronen, S., Zhu, C. Q., Coe, B. P., Liu, N., Watson, S. K., Lam, W. L., & Tsao, M. S. (2006). Large fragment Bst DNA polymerase for whole genome amplification of DNA from formalin-fixed paraffin-embedded tissues. BMC genomics, 7(1), 312.
7 Spits, C., Le Caignec, C., De Rycke, M., Van Haute, L., Van Steirteghem, A., Liebaers, I., & Sermon, K. (2006). Optimization and evaluation of single‐cell whole‐genome multiple displacement amplification. Human mutation, 27(5), 496-503.
2. Loop-mediated isothermal amplification(LAMP)
LAMP was first invented by Tsugunori in 2000, which uses Bst DNA polymerase and a set of primers targeting distinct sites on the template to amplify target DNA. It is the most popular and commercially available detection kit for the diagnosis of tuberculosis. Here is its working mechanism: In the beginning, the inner primer hybridizes to the template to initiate the stretch; then the outer primer binds to its complementary target site and displaces the synthesized single strand DNA, yielding the primary stem-loop DNA. This stem-loop DNA serves as new template for inner primers, which bind to the loop region of the products and continuously displace newly formed DNA strands. The final products appear to be inverted repeats and cauliflower-like structures. LAMP generates extremely large amounts of DNA, about 10^9 copies of target DNA within an hour. Later Eiken et al. introduced additional loop primers to accelerate the reaction, with amplification time less than half that of the original LAMP method.
LAMP can be used to detect various microorganisms, such as E.coli as few as 10 copies and HBV as few as 6 copies. As discussed above, LAMP is highly specific by using four to six primers, which bind six to eight sites of the target simultaneously. The final product can be easily analyzed by various means: 1, electrophoresis, ladder-shaped bands will appear on the agarose gel; 2, turbidity measurement, along with progress of the reaction the white precipitate appears, generating turbidity; 3, colorimetric discrimination, fluoresce probe serves as loop probe while cationic polymers (for example PEI) are used to hybridize LAMP products to precipitate final products and generate detectable color upon excitation, metal-sensitive molecules such as calcein with a shift from orange to green, or pH-sensitive dyes such as Phenol red, Cresol red Neutral red that change their color during reaction because of the release of protons and the drop of pH; 4, current response using Hoechst 33258 that binds DNA minor grooves, causing a significant drop in the current.
LAMP eliminates the need for thermal cycling equipment, as well as cost-consuming device and dye. It amplifies DNA with high specificity and rapidity and generates large amounts of DNA products. It is tolerant to inhibitors of PCR such as blood serum, urine; the reaction can be performed without the extraction of sample DNA.
However, there are some limitations for the LAMP. The primer design is complex because it utilizes four to six primers; Bst Large Fragment can catalyze primer-directed DNA synthesis even if there is no DNA template, resulting from the spurious amplification of the primers; the product it could elongate is restricted to less than 500bp. Moreover, the reaction is not suitable for quantitative analyses.
To overcome the problems, we incorporated palindromic sequence in the primer for efficient amplification, reducing the numbers of primers from six to two (Figure 3). In the initial step, primers would anneal to the template, the newly generated products serve as primers mutually, facilitating next round of amplification. The final products would be tens of thousands of tandem repeats of the target sequence.
Figure 3.Our modified isothermal amplification using two primers. The two primers used to amplify target DNA can pair with each other. It generates initial products, in the same with conventional PCR; next, products with complementary sequence serve as primers mutually, allowing continuous amplification and producing a large amount of tandem repeats of the target sequence.
As a proof of concept, we performed an isothermal amplification using our own designed primers:
| 2×Reaction Mix（RM） | 12.5μl |
|---|---|
| Distilled Water（DW） | 9.5μl |
| Bst DNA Polymerase | 1μl |
| Upstream Primer（40μM） | 1μl |
| Downstream Primer（40μM） | 1μl |
| DNA | 2μl |
Incubated at 63℃ or 60℃ for 60min; then 80℃ for 10min to deactivate Bst DNA Polymerase.
As shown in Figure 4, only the positive template generates ladder-like bands (a, b), which is an evidence that our primers work as expected. When the amplification products were subjected to our PC Reporter system, we obtained a significantly positive signal (c), demonstrating that our modified LAMP method really works.
Figure 4.Amplification using our modified LAMP method. (a), we could amplify DNA targets in different length; Lane 1 for 200 bp, Lane 4 for 400 bp, Lane 7 for 700 bp, and Lane 10 for 1000 bp; Lane 2, 5, 8, 11 and 14 for DNA negative control; Lane 3, 6, 9, and 12 for H2O negative control; Lane 13 for target DNA. (b), we reproduced the amplification using different incubation temperatures; 60 ℃ for 1~9, 63℃ for 10~18. The product length is 200 bp for Lane 1/10, 400 bp for Lane 4/13 and 1000 bp for Lane 7/16. The left 2 lanes are negative control. (c), detection of isothermal products using our PC reporter, we got a significantly positive signal even when the products was diluted by 5 fold (5× dilution of PCR products); 1×PCR products was directly used for the PC reporter. mismatch target for the testing of PCR products with a non correct template, positive control for the detection of template DNA(1.5nM), negative control for no DNA samples.
Compared with the conventional LAMP, our isothermal amplification method requires only two primers, making it easy for primer design. Besides, the amplification will not constrained by the target length anymore; as shown in Figure 4, it could generate product over 1,000 bp. Combined with our PC reporter system, we could imagine an improvement of sensitivity and reduction of turnover time for the nucleic acid detection in clinical practices.
References:
1 Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., & Hase, T. (2000). Loop-mediated isothermal amplification of DNA. Nucleic acids research, 28(12), e63-e63.2 Nagamine, K., Hase, T., & Notomi, T. (2002). Accelerated reaction by loop-mediated isothermal amplification using loop primers. Molecular and cellular probes, 16(3), 223-229.
3 Mori, Y., Nagamine, K., Tomita, N., & Notomi, T. (2001). Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochemical and biophysical research communications, 289(1), 150-154.
4 Mori, Y., Hirano, T., & Notomi, T. (2006). Sequence specific visual detection of LAMP reactions by addition of cationic polymers. BMC biotechnology, 6(1), 3.
5 Tomita, N., Mori, Y., Kanda, H., & Notomi, T. (2008). Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nature protocols, 3(5), 877-882.
6 Tanner, N. A., Zhang, Y., & Evans Jr, T. C. (2015). Visual detection of isothermal nucleic acid amplification using pH-sensitive dyes. LAMP, 5, 6.
7 Safavieh, M., Ahmed, M. U., Tolba, M., & Zourob, M. (2012). Microfluidic electrochemical assay for rapid detection and quantification of Escherichia coli. Biosensors and Bioelectronics, 31(1), 523-528.
8 Hill, J., Beriwal, S., Chandra, I., Paul, V. K., Kapil, A., Singh, T., ... & Vats, A. (2008). Loop-mediated isothermal amplification assay for rapid detection of common strains of Escherichia coli. Journal of clinical microbiology, 46(8), 2800-2804.
9 Poon, L. L., Wong, B. W., Ma, E. H., Chan, K. H., Chow, L. M., Abeyewickreme, W., ... & Peiris, J. M. (2006). Sensitive and inexpensive molecular test for falciparum malaria: detecting Plasmodium falciparum DNA directly from heat-treated blood by loop-mediated isothermal amplification. Clinical chemistry, 52(2), 303-306.
10 Lage, J. M., Leamon, J. H., Pejovic, T., Hamann, S., Lacey, M., Dillon, D., ... & Lizardi, P. M. (2003). Whole genome analysis of genetic alterations in small DNA samples using hyperbranched strand displacement amplification and array–CGH.Genome Research, 13(2), 294-307
Q: Could you please introduce current methods in diagnosis of TB?
A: With the development of molecular biology, various detecting methods have greatly improved our understanding of the biology of the mycobacteria and provide powerful tools to combat the diseases caused by these pathogens. For tuberculosis, different methods have different roles in diagnosis of TB.
PCR-based test has been applied to detection of TB since 1990s, allowing detection of small numbers of disease organisms. However, it shows vulnerability to contamination of samples, and the rate of false-positive is high. With the emergence of advanced techniques, things got better since 2000.
The Real-time Quantitative Fluorescence PCR is one of the most powerful and sensitive techniques available. In 2005, German scientists utilize linear PCR to detect MTB and rifampicin resistance at the same time. After that, the Xpert MTB/RIF developed by Cepheid Company was endorsed by WHO for use, and declared as a major milestone for global TB diagnosis for its accuracy, sensitivity and high detecting speed (two hours per sample). It should be mentioned that the melting curve method developed by Xiamen University has gone through the 6th approval of SFDA, which can detect resistance of rifampicin, isoniazid and ethambutol simultaneously. LAMP (Loop-mediated isothermal amplification) also made the headlines recently.
Q: We noticed that the applications of these advanced detecting tools you mentioned are restricted in clinical practice, why does it happen?
A: Getting research into practice is extraordinarily convoluted and difficult. What has been learned in the research setting often is not implemented into daily clinical practices, even for national institutes of TB control.
One of the main reasons is the high cost of diagnosis. Expense on reagents and maintenance of facilities raises the price to an extremely high level – about ten times higher than traditional detection methods, not to mention the manpower and other resources which have been put into. Expense of TB examination using new methods is not covered by health assurance, which made it more difficult to be accepted by ordinary people.
Another important reason is the preconceived notions. Most of the doctors in China were born in 1970s, and receive traditional medical education. They tend to believe stained bacteria under the microscope and colonies on the culture medium, instead of numbers or curves on an electronic screen.
Q: What’s your perspective on the future of nucleic acid detection?
A: It makes me thrilled. I believe that this tool will unlock a new era of pathogen detection in no more than five years. There will be one day that we are able to diagnose TB with nucleic acid detection even if all other test results are negative. Compared with nucleic acid detection, current diagnostic methods are far from perfect, considering their sensitivity, specificity and time-cost. As to high cost of nucleic acid detection and conventional mind I have mentioned, I believe that time will change everything.
Q: What the necessary features do you expect in our detecting device?
A: I think these features should be included:
1) Able to lyse bacteria and release nucleic acid.
2) Able to amplify the nucleic acid fragment.
3) Able to show the result intuitively.
4) Disposable.
5) Low Cost (lower than 100 yuan for an examination)
6) High specificity and sensitivity.
Q: Let us suppose that a patient wants to know whether he has recovered from TB after treatment, however, both the living and dead bacterium will lead to a positive result. What’s your opinion on this case? Do you think it will cause misdiagnosis?
A: I don’t think it really matters. Note that as a chronic disease, TB has a slow progression and long treatment time. Let’s go into some more details. Patients usually receive more than six months of treatments, 80%-85% of whom will have been recovered in the fourth month. Regarding that it is almost impossible for anyone to expel dead bacteria for two months, which means that, if a patient still has a positive result after six months of treatment, he is not recovered and needs more treatment.
Interview with Director DING Beichuan
Q: From the prospects of laboratory workers in the field of TB diagnosis, do you know how the current TB diagnosis methods apply in the clinical practice?
A: At present in Beijing, all the institutes of TB control are able to diagnose tuberculosis through almost all of the current methods. To be specific, sputum smear and sputum bacteria culture were used 110 000 and 80 000 times last year respectively. It is a striking contrast that molecular detection methods are totally used 5 000 times only. Therefore, classic TB diagnosis methods including sputum smear and bacteria culture play a dominating role in the clinical practice, which becomes an obstacle for promoting these molecular detecting methods.
Situation can be worse in the other provinces of China with little funds investment. In most institutes for TB control in the county level, there are no molecular detecting platforms, not to mention the applications in clinical practice. It is an obvious fact that the proportion of molecular detecting methods is much lower than that in Beijing.
Q: What do you think of molecular detecting methods based on nucleic acid in the future?
A: I think the poor applications of nucleic acid detection methods can soon be changed as the TB diagnostic criteria will be amended with the results of nucleic acid assays being included. At the appointed time, its applications must have a great improvement. After all, nucleic acid detection has its excellent superiorities over the classic diagnostic methods. In conclusion, this kind of method has huge potential to be the first choice for patients suspected of tuberculosis.
Q: If we develop a new nucleic acid detection method or toolkit, which features are necessary for its popularization and employment?
A: According to the national stipulation, it must be sensitive and specific reaching a floor level of 85%. As lab operators, our requirements for detecting time may be different from the doctors and the patients. We just hope that our operational process is as simple as possible, without regard to a short detecting time. For instance, a method with a long waiting time is better than one which requires a short but complex operational time for the laboratory workers. However, from the point of TB patients, a quick detection method is obviously better.
A visit to the Central Lab of Beijing Research Institute for Tuberculosis Control. The director was showing the experiment of drug-resistance testing.
Human Practice group with Director Ding in the Central Lab
Visit to primary medical units
TB mainly occurs in the middle-sized and small cities and rural area in China. In order to have a comprehensive understanding of the current situation, our team went to Zhenghe, FuJian Provence to conduct some interview and collect some data. The county hospital and Zhenghe research institute for tuberculosis control were our destination.
Destination 1: Zhenghe County Hosiptal.
We were luckily to have the opportunity to interview the director of infectious disease department, let’s have a look at the impressive interview.
Q: What are the current diagnostic methods in Zhenghe county? What about the confirmation criteria?
A: The mainstream diagnostic methods in prefecture-level hospitals are sputum smear, sputum culture, and imaging examination, of which bacteria culture is the golden standard for TB confirmation. Also, the clinical symptom is a vital reference for the diagnosis. Unfortunately, the nucleic acid detection tools haven’t been recommended and used.
Q: What’s your opinion of the current examination’s shortcomings?
A: For the bacteria culture, the main deficiency is high time cost and low positive rate (no more than 20%). It needs to emphasize that the bacteriology diagnosis of MDR-TB, which is more and more common, needs almost twice the time. For the imaging examination, its application is too limited which means it can only be used for the pulmonary tuberculosis, not extra pulmonary tuberculosis. Meanwhile, the result of imaging examination can’t confirm the TB, because of the several diseases having similar imaging result.
A visit to the Infectious Disease Department in Zhenghe County Hospital
Q: Is there any problem of TB control in Zhenghe County?
A: On the one hand, the base installation of TB control and expenditure are insufficient, leading to the large gulf between the primary medicine units and metropolis medicine units. We also need more professional and technical practitioners to provide professional service and promote the development of primary medicine care. By the way, the communication between institute of TB control and hospitals is supposed to be more thorough to stop passing the buck to each other.
4. Have you ever heard of the nucleic acid test and would you like to use it?
Director: There are no such methods or tools (nucleic acid detection) in either hospital or institute of TB control in Zhenghe County and we know little about that. If there is a new tool with high specificity, high sensitivity and low cost, it will surely make a great contribution to our hospital.
Destination 2: Zhenghe research institute for tuberculosis control.
We were warmly welcomed by the director of epidemic prevention station, the conversation with him give us lots of inspiration.
Q: What is the current situation of tuberculosis in Zhenghe County, and what are the demographic characteristics of TB patients?
A: At present, Zhenghe County has a population of over 200 000 and there are 170 000 people permanent living here. Among the whole population, about 80 people are tuberculosis patients. TB was a serious disease during the 1990s. It was controlled a few years ago with an ascending trend in the incidence rate. The prevalence in male was higher than in female, and gradually increases by age, with most of the patients between 18 to 60 years old, which accords with the national statistics results. However, with large number of peasant-workers going out for jobs, the morbidity in rural areas is consistent with that in cities, which is different from the current situation of the whole nation.
Q: What are the tuberculosis diagnosis procedures and the diagnostic criteria in Zhenghe County?
A: In the current situation, the primary diagnostic methods are PPD test, sputum smear, bacteria culture and chest X-ray, along with the patients’ clinical signs. In addition, we would sometimes use erythrocyte sedimentation rate measurement and reactive protein items. But the most frequently used one is sputum smear. Due to the high price and complicated operations, we usually turn over the sample to the municipal institution to get the finally result.
Q: What are the current control measures for TB in the county epidemic prevention station?
A: All the newborn babies will receive BCG for the early prevention. But the vaccine can only function effectively on pulmonary tuberculosis for 3-4 years. For the suspected cases, we will provide a initial examination freely, but the following examination will be self-paying if the TB is confirmed. Unlike the examination, most of the drugs are free, of which the first-line anti-tuberculosis drugs are all free and the second-line drugs are paid 70% by government.
Q: Is there any problem of TB control in Zhenghe County?
A: The biggest challenge we are facing is the insufficient fund. It’s difficult for us to make ends meet because we never fully funded by the government while the policy make us foot the bills by ourselves. The insufficient fund leads to the shortage of intelligence and professions, which further results in a vicious circle. Withal, many patients can’t accept the self-paying treatment and examination of which the price is a little bit expensive. From my point of view, the backward regional economic and the distrust for medical Institutions lead to the terrible condition.
Our team member with Director Lv of Epidemic Prevention Station in front of Zhenghe Research Institute for Tuberculosis Control