**2.3 Sample preparation – PURE**

The sample processing method is the next important step in molecular diagnostics. Silicabased methods are well known and have been applied to a wide variety of samples, including blood and tissue (Bendall, 2002). However, these methods are unsuitable for resource-limited facilities due to the cumbersome procedures involved, including washing with organic solvents using high-speed centrifugation. Therefore, we have developed a simple and swift sample processing method named PURE. Thus far, it has been confirmed that PURE can be successfully applied to sputum, blood, serum, and swab samples. The mechanism of PURE is described below:


Novel Molecular Diagnostic Platform for Tropical Infectious Diseases 451

Figure 4 shows the overall process of the PURE-LAMP assay system. First, an aliquot of sample is placed in a heating tube and heated at an optimized temperature (70–90 °C) to lyse the target pathogen. Then, the heating tube is attached to an adsorbent tube, and the treated solution is vigorously mixed with adsorbent powder. Next, the injection cap is inserted into the absorbent tube, which is then squeezed to elute the solution containing purified DNA. These processes can be performed in approximately 10 minutes or less for a particular sample. The LAMP reagents deposited in the lid of the tube are reconstituted by shaking the tube several times and then incubating it at around 65 °C for 30–40 minutes. Finally, the results of amplification are detected by simply observing the fluorescence using the LED lights provided in the incubator. As mentioned above, we have successfully developed simple technologies for all the 3 steps required in molecular diagnostics, that is, the PURE method for sample preparation, the LAMP for amplification, and the calcein method for detection. Therefore, a combination of these technologies can be considered as a platform for a new molecular diagnostic tool with

**3. Application of the newly developed platform for diagnosing tropical** 

test is not sufficient to detect the parasites in patients with early-stage malaria.

The developed platform has been applied to the following 3 tropical diseases to evaluate its

Malaria is 1 of the 3 major infectious disease endemics in most tropical countries. More than 500 million people have been infected, and more than 1 million people die from malaria each year, mostly infants and pregnant women. Of the 4 malaria causing species, *Plasmodium falciparum* often causes severe, acute, and fatal malaria. In most developing countries, malaria is confirmed mainly by a blood smear test, although the sensitivity of the

Dried LAMP reagents using *P. falciparum* (Pf)-specific primers and pan genus (Pg) primers were developed in this study. The Pg LAMP primers were designed on the basis of the homogeneous sequence shared by all the 4 malaria species, thus providing the same primer specificity for all the 4 species. The Pf-specific LAMP primers were designed based on mutations between the Pf sequence and the other 3 sequences, making the primer specific only to *P. falciparum*. If both Pf and Pg LAMP assays give positive results, it can be interpreted that the patient is infected by *P. falciparum*. On the other hand, if only the Pg LAMP assay gives positive results, the patient can be diagnosed with malaria caused by 1 or

The sensitivity of PURE and malaria-LAMP have been evaluated by using of cultured *P. falciparum* parasites obtained from the American Type Culture Collection (ATCC). As shown in figure 5, both Pf and Pg malaria-LAMP assays can detect down to 1 parasite in 1 μl blood, processed by the PURE method. This sensitivity of 1 parasite/μl blood is much higher than that of smear microscopy test (~50 parasites/μl blood for routine tests in an endemic area (Moody, 2002)). It has been reported that the initial malarial symptoms appear after the accumulation of approximately 1,000 parasites/ml of blood (Andrews et al., 2005). Therefore, PURE-malaria-LAMP is sensitive enough to detect parasites in patients who

Human African trypanosomiasis (HAT) is one of the most neglected disease endemics in central African countries (Hotez, 2007). HAT is caused by an infection of protozoa

the desired simplicity.

performance as a practical diagnostic system.

more of the other 3 malarial parasites.

present with the initial symptoms of malaria.

**3.1 Malaria and human African trypanosomiasis** 

**diseases** 

Fig. 3. Performance of the PURE–LAMP system applied for blood processing A) Pictures of the blood sample solution (3.6% blood in an alkaline-based extraction solution) before and after PURE treatment. Left, Before PURE treatment; Centre, After PURE treatment; Right, distilled water (reference).

B) LAMP kinetics obtained by real-time turbidimetry for the 3 sample solutions with 1,000 copies of a template DNA spiked prior to PURE treatment.Green line, Before PURE treatment (directly added the solution to LAMP reaction); Red line, after PURE treatment; Blue line, control (distilled water); Purple line, negative control.

Figure 3 shows the performance of the PURE method applied for blood processing. An aliquot of blood was mixed with the extraction solution and heated at 70 °C for 5 minutes. Almost colorless solutions have been obtained by mixing the solution with the adsorbent powder (Fig. 3-A). The graph of real-time turbidimetry (Mori et al., 2004) in figure 3-B shows the LAMP kinetics for the 3 samples using 1,000 copies of a spiked template DNA. Untreated blood samples did not provide a positive reaction due to the inhibition from blood and extraction solution. However, PURE-treated blood samples showed almost the same kinetics as those of distilled water, indicating that PURE can remove inhibitory materials quite effectively from the blood samples without any loss of DNA.

Fig. 3. Performance of the PURE–LAMP system applied for blood processing A) Pictures of the blood sample solution (3.6% blood in an alkaline-based extraction

materials quite effectively from the blood samples without any loss of DNA.

Blue line, control (distilled water); Purple line, negative control.

treatment; Right, distilled water (reference).

solution) before and after PURE treatment. Left, Before PURE treatment; Centre, After PURE

Figure 3 shows the performance of the PURE method applied for blood processing. An aliquot of blood was mixed with the extraction solution and heated at 70 °C for 5 minutes. Almost colorless solutions have been obtained by mixing the solution with the adsorbent powder (Fig. 3-A). The graph of real-time turbidimetry (Mori et al., 2004) in figure 3-B shows the LAMP kinetics for the 3 samples using 1,000 copies of a spiked template DNA. Untreated blood samples did not provide a positive reaction due to the inhibition from blood and extraction solution. However, PURE-treated blood samples showed almost the same kinetics as those of distilled water, indicating that PURE can remove inhibitory

B) LAMP kinetics obtained by real-time turbidimetry for the 3 sample solutions with 1,000 copies of a template DNA spiked prior to PURE treatment.Green line, Before PURE treatment (directly added the solution to LAMP reaction); Red line, after PURE treatment;

Figure 4 shows the overall process of the PURE-LAMP assay system. First, an aliquot of sample is placed in a heating tube and heated at an optimized temperature (70–90 °C) to lyse the target pathogen. Then, the heating tube is attached to an adsorbent tube, and the treated solution is vigorously mixed with adsorbent powder. Next, the injection cap is inserted into the absorbent tube, which is then squeezed to elute the solution containing purified DNA. These processes can be performed in approximately 10 minutes or less for a particular sample. The LAMP reagents deposited in the lid of the tube are reconstituted by shaking the tube several times and then incubating it at around 65 °C for 30–40 minutes. Finally, the results of amplification are detected by simply observing the fluorescence using the LED lights provided in the incubator. As mentioned above, we have successfully developed simple technologies for all the 3 steps required in molecular diagnostics, that is, the PURE method for sample preparation, the LAMP for amplification, and the calcein method for detection. Therefore, a combination of these technologies can be considered as a platform for a new molecular diagnostic tool with the desired simplicity.
