Comparison of Purity and Concentration Values of Mycobacterium tuberculosis DNA Extraction Result from the Boiling and Spin Column Method

The Polymerase Chain Reaction (PCR) technique is extensively employed in molecular biology to precisely detect Mycobacterium tuberculosis . Prior to conducting PCR, extracting of high-quality genomic Deoxyribonucleic Acid (DNA) is crucial to ensure accurate and reliable results. The primary objective of this study is to conduct a comparative analysis of the purity and concentration of M. tuberculosis DNA acquired through the utilization of the boiling method and the spin column extraction methods. A descriptive comparative research design was employed, utilizing a sample of 16 sputum specimens that had previously been confirmed as positive for M. tuberculosis through Acid-Fast Bacteria (AFB) examination and Molecular Rapid Test (MRT). The extraction of DNA was carried out using the boiling method and the spin column method. Subsequently, the concentration and purity of the extracted DNA were assessed using the NanoDrop Spectrophotometer, and the results were compared. The obtained yield of M. tuberculosis DNA isolates through the boiling method ranged from 9.6 ng/µL to 1258.7 ng/µL, with an average purity value of 1.23. Conversely, for the spin column method, the concentration of M. tuberculosis DNA isolates ranged from 8.7 ng/µL to 207.8 ng/µL, with an average purity value of 1.83. In conclusion, there is a significant difference between the purity and concentration of M. tuberculosis DNA extraction results using the boiling method and spin column methods

PCR is a method recommended by the WHO for of examining M. tuberculosis infection due to its high sensitivity and specificity (8).
Prior to conducting identification via The spin column method resulted in an average DNA concentration of 5.93 ± 0.94 µg/mL and an average purity of 1.69 ± 0.09.
The purity value is lower than the modified CTAB method results, which is 1.73 ± 0.

MATERIALS AND METHODS
This μL of W1 buffer was added and centrifuged at 14,000 x 16,000 g for 30 seconds.
following this, 600 μL of Wash buffer was added and centrifuged at 14,000 x 16,000 g for 30 seconds.In order to desiccate the column matrix, the sample mixture was centrifuged at 14,000 x 16,000 g for 3 minutes, followed by the addition of RNase-Free Water to elute the DNA.The DNA isolates were then subjected to concentration and purity measurement using the NanoDrop Spectrophotometer.

RESULTS
The data obtained from the measurements using a NanoDrop Spectrophotometer consists of concentrations in ng/µL and purity values obtained from the absorbance ratio at λ 260 nm and 280 nm, as presented in Table 1. 137

DISCUSSION
The DNA extraction processes using the boiling method and the spin column method differ in principles and the reagents used.In the boiling method, the sputum sample is first treated with 4% NaOH and then subjected to health during annual rounds of data collection, in 2022, there were 202 countries and territories reporting data on TB cases, representing more than 99% of the world's population (3).Indonesia stands out as one of the countries contributing to the high increase in TB cases.It ranks third with 824 thousand cases of TB, following India and China, and records 93 thousand TB-related deaths annually, equivalent to 11 deaths per hour (4).The government's initiatives to reduce TB cases in Indonesia are outlined in the National Medium-Term Development Plan (RPJMN) for the health sector, spanning from 2020 -2024.These initiatives are directed toward accelerating the elimination of TB by 2030.Hence, there is a pressing need for rapid and highly sensitive laboratory testing to identify TB cases early and ensure optimal treatment.M. tuberculosis is a pathogenic Grampositive bacterium that commonly infects the lungs.It can also infect other organ systems, such as the gastrointestinal, musculoskeletal, lymphoreticular, skin, liver, and reproductive systems (5,6).The Polymerase Chain Reaction (PCR) method is one of the molecular techniques used to detect M. tuberculosis, alongside microscopic examination and bacterial culture.The PCR technique exhibits very high sensitivity in detecting PCR, it is essential to extract genomic DNA in order to obtain pure DNA.This extracted DNA serves as a template during the PCR amplification process.Therefore, the quality of the extracted DNA becomes one of the factors that can influence the success of the PCR examination.Quantitative and qualitative tests of the isolated DNA are performed to assess its concentration and purity using a spectrophotometer (9).Optimal DNA purity is indicated by a λ 260/280 ratio between 1.8 to 2.0, reflecting high-quality standards (10).There are several methods for extracting M. tuberculosis DNA, including conventional and kit-based extraction methods.The boiling method is a conventional DNA extraction method 135 that is simple, cost-effective, fast, and capable of extracting bacterial DNA from Gram-positive and Gram-negative bacteria at high temperatures.It has been proven to be efficient (11).According to research conducted by Fihiruddin et al. (8), the boiling method with heating at 95°C for 5 minutes was found to be the optimal temperature for DNA extraction from sputum samples.This study revealed an average DNA concentration of 0.94 ng/μL and a purity value of 1.23 for M. tuberculosis DNA using the boiling method at 95°C (8).The solid-phase extraction method, also known as the spin column extraction, utilizes a silica membrane as the nucleic acid binder in the DNA extraction process.This method employs a kit, and the nucleic acid binding is optimized with specific buffer solutions, pH, and appropriate salt concentrations.Using silica particles in the spin column method ensures safety and minimizes the potential for cross-contamination (12,13).According to the research by Ndhlovu et al. (13), it was demonstrated that M. tuberculosis DNA was successfully extracted from sputum samples using the spin column kit method, and it outperformed the modified Cetyl trimethylammonium bromide (CTAB) extraction method.
study employs a descriptive comparative research design, specifically utilizing a Post-test Only Control Group Design.Sixteen sputum samples were collected from patients diagnosed positive for Acid-Fast Bacilli (AFB) through microscopic examination and M. tuberculosis through the Molecular Rapid Test (MRT) from hospitals in the city of Cimahi, using purposive sampling.The research was conducted at the 136 Biosafety Level 2 Laboratory, Department of Medical Laboratory Technology, Health Polytechnic Kemenkes Bandung, during May and June 2023.The sputum samples collected from tuberculosis patients in sputum pots underwent a washing process using a 1:1 ratio of 4% NaOH as a sputum dilution solution to removed mucus from the samples (15).The resulting sample mixture was vortexed at 2,500 rpm for 20 seconds and then centrifuged at 13,000 x 16,000 g for 2 minutes, followed by the removal of the supernatant.The DNA extraction using the boiling method was conducted by pipetting 200 μL of processed sputum sample into a microtube, followed by the addition of 100 μL of 1x TE buffer and one μL of Proteinase K.The mixture was then frozen at -18°C for a minimum of one hour, heated at 60° C for one hour, and boiled for 5 minutes at 100°C.Subsequently, the resulting DNA extraction was centrifuged for 3 minutes, and the supernatant was transferred to a new microtube.The obtained DNA isolate was then analyzed for concentration and purity using the NanoDrop Spectrophotometer.The DNA extraction using the spin column method was carried out with the Geneaid Viral Nucleic Acid Extraction Kit II (VR300).Initially, 200 μL of processed sputum sample was pipetted into a microtube, followed by 400 μL of VB lysis buffer.The mixture was vortexed and incubated for 10 minutes.Subsequently, 450 μL of AD buffer was added and vigorously shaken.The lysate mixture was transferred into the silica column VB column (600 μL), and centrifuged at 14,000 x 16,000 g for one minute.The liquid passing through the silica membrane was discarded, and the VB column was placed back into the collection tube.The remaining lysate mixture was added back into the VB column and centrifuged at 14,000 x 16,000 g, with the liquid in the collection tube being discarded.Then, the VB column was transferred to a new collection tube, and 400 The obtained data underwent rigorous comparative statistical analysis to discern any disparities in the purity and concentration values of DNA extraction results between the two distinct extraction methods.Initially, the sign test was conducted to compare the purity of the boiling method with the purity of the spin column method.The results suggest that the Sig value for DNA purity between the boiling method and the spin column method was < 0.05.It indicates a highly significant difference in purity between the Boiling Method and the Spin Column Method.This value suggests that the two methods produce significantly different levels of purity.The Wilcoxon test was conducted to analyze the concentration of the Boiling Method in comparison to the concentration of the Spin Column Method.The results indicate that Z-score: -3.067b and Asymptotic Significance (2-tailed): 0.002.With a significance level (p-value) of 0.002 (< 0.05.), the results suggest a statistically significant difference between the concentrations obtained through the Boiling Method and the Spin Column Method.The negative Z-score (-3.067) indicates that the concentration of the Boiling Method is lower compared to the Spin Column Method.
TE buffer (Tris-EDTA) solution, which aids maintaining the solution's pH, dissolving the extracted DNA, and protecting it from degradation during storage (16).The addition of proteinase K 139 serves to degrade proteins in the nucleic acid.Cooling at -18°C and subsequent heating at 60°C and 100°C are intended to disrupt bacterial cells and tissues through physical means.Following this, the centrifugation process separates DNA, causing proteins, lipids, and other components, excluding DNA, to precipitated, thus enabling DNA concentration in the supernatant.The supernatant is then carefully transferred to a new sterile microtube.The addition of centrifugation in the final step of the boiling method is a modified procedure performed by researchers following the DNA extraction procedure of the boiling method described byNurhayati and Darmawanti(17), as well as in the study conducted by Fihiruddin et al.(8).The DNA extraction process using the spin column method is systematically executed in a microtube equipped with a silica membrane column(13).Reagents such as VB lysis buffer and AD lysis buffer are intriduced to facilitate the lyse of bacterial cell walls, enabling the release of cellular components.The lysis buffer solution serves to break down tissues, disrupt cell membranes, and eliminate contaminants, thus facilitating the extraction of genomic DNA.The addition of W1 buffer and Wash buffer serves the purpose of eliminating any residual proteins that might be attached to the DNA and eliminating prior buffer salts (18).Each addition of the reagents is followed by centrifugation, which applies centrifugal force to aid in the binding of DNA to the silica matrix, causing cellular components other than DNA to flow to the bottom of the tube.Subsequently, DNA is eluted using RNAse-free water to release the DNA bound to the silica matrix and collect it at the bottom of the tube.Additionally, RNAse-free water protects nucleic acids from degradation by nucleases, such as DNase or RNase (19).The extracted DNA samples are stored at a stable temperature of -20°C before being subjected to measurements using the NanoDrop spectrophotometer (18).The conducted research highlighted a notable disparity between the purity and concentration of DNA obtained from M. tuberculosis using the boiling and spin column methods.Among the sixteen data points gathered from the purity results of DNA extracted using the boiling method from M. tuberculosis, only one sample (6.25%) fell within the range of pure values, specifically sample number 15.The average purity value of the extracted DNA from M. tuberculosis using the boiling method was 1.23.Conversely, from the data acquired in the purity results of DNA using the spin column method, 15 samples (93.75%) fell within the range of pure values, specifically 1.8 -2.0.The average purity value of the extracted DNA from M. tuberculosis using the spin column method was 1.83.This observed purity outcome is line with a prior study by Fihirudin et al. (8), where the purity 140 values of DNA extracted using the boiling method at various temperatures (85°C, 90°C, and 95°C) were within the range of 1.18 -1.23.Significantly low purity results were obtained in the samples from the DNA purity measurements using the boiling method (samples number 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, and 16).This outcome may be attributed to the presence of various contaminants such as proteins, lipids, carbohydrates, and other cellular components in the extraction results, leading to compromised DNA purity (20,21).Notably, DNA and RNA are absorbed at λ 260 nm, while contaminants, including proteins and phenol, exhibit strongly absorbtion at λ 280 nm (22,23).Consequently, the heightened absorption of proteins at λ 280 nm can significantly impact DNA purity, as the assessment of DNA purity is reliant on the absorption ratio at λ 260/280 nm.The elevated levels of the contaminants ultimately contribute to diminished DNA purity.In the boiling extraction method, a particular step was altered to isolate DNA from contaminants or other components through a single centrifugation process follwong heating.Unlike certain other extraction methods, the boiling method does not incorporate washing and purification steps within the extraction process.This particular aspect presents a notable limitation of the method, potentially allowing contaminants to persist within the DNA extraction sample (24).Regarding the sample of DNA isolate obtained through the boiling method, sample number 8 exhibited measurement errors in both concentration and purity assessments.Such error could potentially be attributed to several factors, including the sample possessing an absorbance value that falls beyond the detection range of the NanoDrop spectrophotometer instrument.The quality of the extracted DNA isolates from M. tuberculosis samples, which remained visibly concentrated and presented a reddish-brown color (likely due to the inclusion of sputum samples mixed with blood), may indicate the presence of contaminants.These contaminants can significantly interfere with the absorption of light transmitted through the sample during measurement, resulting in the inability to accurately detect absorbance values required for determining concentration and purity at specific wavelengths, thereby leading to errorneous results.Notably, the spectrophotometer instrument is designed with upper detection limits for dsDNA up to 3700 ng/µL, ssDNA up to 2400 ng/µL, and RNA up to 3000 ng/µL.In instances where the sample color is excessively concentrated, it may lead to concentrations surpassing the instrument's detection limits, ultimately resulting in errorprone measurement outcomes.Moreover, as outlined in the NanoDrop spectrophotometer 141 user manual, the lack of absorbance results can also be attributed to the sample or aliquot's lack of homogenity.Hence, it is imperative to ensure the homogenization of the DNA isolate sample before conducting measurements using the instrument to mitigate the likelihood of generating errorneous results (25).Within the sample from the purity measurement of DNA using the spin column method, sample number 11 exhibited a notably low purity value.Such an outcome could be attributed to the presence of protein contaminants and other impurities within the extracted DNA isolate.Notably, the DNA extraction process carried out using the spin column method encompasses several intricate steps, including cell wall lysis, the separation of DNA from impurities and other cellular components, DNA binding to the silica column, as well as DNA washing and purification stages.While the utilization of the spin column method represents a more comprehensive process compared to the boiling method, it does not entirely preclude the possibility of the extracted DNA via the spin column method yielding lower purity levels.This potential disparity could arise from various factors, including potential errors during the analytical stages, inaccuracies during pipetting, and technical oversights during the measurements conducted using NanoDrop spectrophotometer.Upon analyzing the samples using the boiling method for DNA concentration, notably higher concentration values were observed in the samples 1, 2, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, and 16.However, these samples exhibited comparatively lower purity values in contract to the DNA concentration results acquired through the spin column method.Similarly, in a particular case of an isolate obtained through the spin column DNA extraction method, specifically sample number 11.There was an observed high concentration value accompanied by a low purity value.This particular scenarion may be attributed to the presence of an additional type of nucleic acid, namely single-stranded DNA (ssDNA), within the sample, which was measured at λ 260 nm.As a result, it led to elevated DNA concentration (22).Another form of nucleic acid that can be measured at λ 260 nm is RNA.However, if RNA contamination were presenst, the resulting purity values would exceed >2.Since there were no samples with purity values >2 from the purity measurements, the likelihood is that the measurable nucleic acid is ssDNA (single-stranded DNA).The presence of such contamination can significantly contribute to an increase in DNA concentration results.Notably, one of the potential contaminants impacting the samples could be the presence of DNA from other genomes, which might inadvertently 142 carry over through reagents, equipment, or various laboratory conditions.Therefore, it is crucial to pay attention to these factors.Additionally, the implementation of stringent controls measures is vital to ensure the integrity of the work conducted.The DNA extraction results obtained using the spin column method from samples 3, 6, 10, 13, and 14 yielded high concentrations (>50 ng/µL) along with purity values falling within the range of 1.8 -2.0.This outcome can be attributed to the efficient extraction of DNA molecules in substansial quantities, ensuring minimal contamination from proteins other impurities.In the measurement of DNA extraction results using the spin column method, there are several samples, namely samples number 1, 2, 4, 5, 7, 8, 9, 12, 15, and 16, which exhibit low DNA concentrations (<50 ng/µL), but possess a high level of purity.The diminished DNA concentration in these instances can be linked to various factors, such as suboptimal processes related to cell wall lysis and the separation of DNA from cellular tissues, ultimately resulting in a limited amount of extracted DNA.Additionally, incomplete DNA binding during the DNA binding stage may contribute to an insufficient DNA yield.The likelihood of compromised DNA purity during measurements using the NanoDrop Spectrophotometer can be attributed to inadequate sample homogenization prior to the measurement process.The fundamental principle of spectrophotometry stipulates that the utilized sample should be clear and fully dissolved (22,26).Another influential factor impacting the magnitude of DNA extraction concentration is the composition of the added lysis buffer, the specific physical treatment employed in each extraction method, as well as the buffer's capacity utilized in the DNA extraction process to effectively disintegrate bacterial cells (24).CONCLUSIONS The conducted research findings clearly delineate a significant disparity in the purity of M. tuberculosis DNA when employing the boiling method versus the spin column method.Specifically, the DNA purity attained from the boiling method is comparatively lower, with a recorded DNA purity level of 1.23.In contracts, the employment of the spin column method led to a notably higher DNA purity level of 1.83, aligning more closely with the average range value of 1.8 -2.0.Discernible differences exist in the concentration values of M. tuberculosis DNA between the boiling method and the spin column method.Specifically, the concentration of M. tuberculosis DNA using the boiling method ranged from 9.6 ng/µL to 1258.7 ng/µL.In contrast, the utilization of the spin column method led to a concentration range for M. 143 tuberculosis DNA spanning from 8.7 ng/µL to 207.0 ng/µL.Moving forward, it is recommended that further research should involve conducting PCR with M. tuberculosis specific primers, thereby enabling the visualization of DNA bands with a basepair length that matches the target gene of M. tuberculosis.Moreover, stringent control checks are imperative to ensure the accuracy of the extraction procedures and complete sterility of the utilized tools, thereby mitigating the occurrence of potential contaminations that could otherwise impact the concentration value and purity of the resulting DNA. 14.

Table 1 .
Data of Measurement Results for Concentrationa Purity of M. tuberculosis DNA Extraction Using Boiling Method and Spin Column Method

No Sample Positivity DNA Extraction Boiling Method DNA Extraction Spin Column Method Purity Value Concentration Value (ng/µL) Purity Value Concentration Value (ng/µL)
11, 13, 14), and positive M. tuberculosis sputum samples confirmed by the Molecular Rapid Test (samples number 8, 10, 12, 15, 16).The boiling method yielded a higher DNA concentration compared to the spin column method, ranging from 9.6 ng/µL to 1258.7 ng/µL for the boiling method, and from 8.7 ng/µL to 207.8 ng/µL for the spin column method.However, an error was encountered when measuring the concentration and purity of the M.tuberculosis DNA isolates using the boiling method for sample number 8.Analysis of the measurement results forM.tuberculosisDNAconcentration using the boiling method, as outlined in Table1,highlightssample number 3 as having the lowest DNA concentration.Conversely, the application of the spin column method reveals that samples number 1, 2, 4, 5, 7, 8, 9, 12, 15, and 16 present notably low DNA concentrations.Moreover, examination of the results from the DNA purity measurements in M.tuberculosis using the boiling method, illustrated in Table1

Table 2 .
Results of DNA Purity Data Processing in M. tuberculosis Using the Boiling Method and the Spin Column Method . In Table 1, indicates that samples 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, and 16 exhibited relatively low DNA138 purity values (with sample 8 resulting in an error).In contrast, the DNA purity measurements of M. tuberculosis using the spin column method reveal that only sample 11 demonstrates a notably low DNA purity value.