Optimization of nucleic acid extraction methods for rapid detection in pandemic situations or diseases with high prevalence

Graphical abstract

Highlights

• •Heat-based NAE method could easily be applied in laboratory without centrifugation and freezing equipment.
• •Speed variation is crucial parameter to increase efficiency of magnetic beads-based NAE method.
• •Heating and speed actuator equipment is important system for constructing magnetic beads-based local extraction machine.

Nucleic acid extraction (NAE) is an essential component of many assays used for diagnostic and research purposes. During a pandemic, performing NAE is associated with significant challenges due to material shortages, high costs, limited laboratory facilities, and the time required. This delays patient diagnosis and treatment, particularly in countries that do not produce NAE devices. Here, we tested several procedures to speed up the extraction process using common laboratory reagents, finding that heat-based nucleic acid extraction of severe acute respiratory syndrome coronavirus 2 (SARS-COV2) is a detection method that can be easily implemented in laboratories, even those without centrifugation facilities. Additionally, the magnetic beads-based extraction method does not require a heating step to isolate RNA. We noted that speed variation during mixing is required to increase extraction efficiency. These results can be considered when designing local machines and simplifying extraction processes to overcome the bottlenecks associated with clinical diagnostics for large samples.

Given the importance of rapid clinical diagnoses, World Health Organization (WHO) has recommended reverse-transcriptase polymerase chain reaction (RT-PCR) as the gold standard diagnostic method for several diseases. NAE is a crucial step in RT-PCR analysis, which is used for molecular research, diagnostic testing, downstream analysis (i.e., PCR, cDNA synthesis, and RNA/DNA sequencing), and identifying the most effective treatment for microbial infections, such as diseases caused by bacteria (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, or Acinetobacter baumannii), viral infections (i.e., SARS-CoV-2, human immunodeficiency virus (HIV), hepatitis B virus (HBV), dengue, and zika), and other diseases like malaria and cancer.

The NAE method typically involves cell lysis, separating nucleic acid from cell debris, and nucleic acid purification. The column-based technique has traditionally been the most common NAE method for obtaining high nucleic acid yields [1]. However, researchers have recently moved from column-based to magnetic beads-based NAE, which simplifies the process of NAE and selectively binds nucleic acid to magnetic beads. Magnetic beads-based NAE enables large samples to be analyzed rapidly using automated or semi-automated machines, which is beneficial during pandemics or for high-prevalence diseases. However, it requires expensive devices (e.g., instruments, kits, and consumables), has high operating costs, and is a closed system. Another method is the heat-shock technique, which damages cells to obtain nucleic acids and is qualitatively equivalent to commercial kits [2].

In this study, we obtained nasopharyngeal specimens from Universitas Padjadjaran's C.29 laboratory, which were placed in a Tris-EDTA (TE) buffer solution (10 mM Tris pH-7.4, and 0.1 mM EDTA). The Faculty of Medicine Ethics Committee from the Universitas Padjadjaran approved this study (Approval number: 779/UN6.KEP/EC/2022). We extracted the total RNA using a magnetic beads-based or heat-based extraction method. We heated the samples at 90 °C for 5 min, followed by either centrifugation (8000 rpm for 5 min) or cooling (6 °C for 5 min). The magnetic beads-based extraction was performed using a MagEx RNA Extraction kit (Sinergi Untuk Nusantara-Dena Danar Djaya, Jakarta, Indonesia) or a MagMAX RNA Extraction kit (Thermo Fisher Scientific, Waltham, MA, USA) and a KingFisher Flex System machine with a heating block feature (Thermo Fisher Scientific). The samples were extracted with or without the heating process and with or without speed variation. The samples underwent RT-PCR (AriaMx Real-time PCR System, Agilent, Santa Clara, CA, USA) using the bioPerfectus RT-PCR kit. The ORF1ab, N, and RNAse P genes were used as controls (Covid-19 Coronavirus Real-Time PCR kit, bioPerfectus technologies, Hamburg, Germany).

Typically, heat-based NAE involves three main steps: cell destruction via heat-shock, separation of nucleic acids from cell debris, and nucleic acid isolation. Cell debris is usually separated using centrifugation; however, this step can be replaced with a cooling step to obtain the total nucleic acid in the supernatant [3]. As solids that dissolve in liquids decrease in solubility as the temperature drops, we collected the RNA from the supernatant at room temperature following the heat-shock step without needing a centrifugation or cooling step. Although heat-shock followed by centrifugation demonstrated the lowest cycle threshold (Ct) value, extraction without centrifugation was acceptable, as indicated by the appearance of Ct RNAse P values. US Center for Disease Control and Prevention (CDC) has recommended RNAse P gene as an endogenous internal control for RT-PCR (Table 1).

Tabel 1.

Reverse-transcriptase polymerase chain reaction (RT-PCR) results of RNA extraction parameter optimization (heat-based). Positive results indicated with bold word.

Sample code	Ct ORF1ab	Ct N-gene	Ct RNAseP	Interpretation	Step
A1	19.9	19.62	25.89	Positive	no centrifugation
A2	23.2	22.43	28.88	Positive
A3	–	–	31.42	Negative
A1	18.39	18.32	24.99	Positive	with centrifugation
A2	22.15	21.33	33.35	Positive
A3	–	–	33.43	Negative
A1	19.15	18.68	27.06	Positive	Cooling
A2	22.46	21.45	30.87	Positive
A3	–	–	33.51	Negative

Ct: cycle threshold.

We also designed a magnetic beads-based RNA extraction process by optimizing heating step or speed of magnetic beads motion. The magnetic beads-based method consists of three steps: lysis, wash, and elution. We heated samples in a lysis buffer at 95 °C. During the elution step, we heated the samples at 65 °C. At this temperature, RNA is released from the magnetic rod into the elution buffer. We used an extraction kit that listed a heating step (MagMAX kit) and did not list a heating step (MagEx kit) in its manual. Using the MagEx kit, heating during the lysis and elution stages did not change the Ct values significantly compared to when the heating step was omitted. We observed similar effects on samples extracted using MagMAX, whereby skipping the heating step did not reduce the extraction quality (Table S1).

Numerous experiments have investigated controlling magnetic beads motion to enhance the capture and mixing processes [4]. We investigated NAE using one speed (slow mode) or varied speeds (slow, moderate, and fast modes). Our result showed that varied-speed modes have a higher positivity rate in detecting SARS-CoV2 than the one-speed method. Further, the one-speed mode shifted the Ct value by five larger, indicating lower RNA extraction efficiency than the varied-speed mode (Table 2). This suggests that speed variation in the movements of magnetic rods is required to increase RNA extraction efficiency.

Tabel 2.

Reverse-transcriptase polymerase chain reaction (RT-PCR) results of RNA extraction parameter optimization (magnetic beads-based). Positive results indicated with bold word.

	Sample code	Ct ORF1ab	Ct N-gene	Ct RNAseP	Interpretation	Step
	S1	37,11	36,36	29,81	Positive	Slow Mode
	S2	-	38,84	-	Positive
	S3	39,22	38,2	34,07	Positive
	S4	–	–	36,98	Negative
	S5	39,36	39,72	32,3	Positive
	S6	-	37,78	30,83	Positive
	S7	38,85	34,81	30,73	Positive
	S8	39,02	35,93	29	Positive
	S9	–	–	28,82	Negative
	S10	39,19	35,05	27,95	Positive
	S11	–	–	33,22	Negative
	S12	–	–	34,29	Negative
	S13	–	–	32,19	Negative
	S14	–	–	–	Invalid
	S15	–	–	31,92	Negative
	S16	–	–	32,88	Negative
	S17	–	–	30,4	Negative
	S18	–	–	30,59	Negative
	S19	–	–	33,11	Negative
	S20	–	–	30,4	Negative
	S1	32,09	30,8	28,48	Positive	Slow, moderate and fast modes
	S2	35,9	33,69	27,68	Positive
	S3	34,12	33,3	27,71	Positive
	S4	35,46	32,58	31,46	Positive
	S5	35,34	33,52	29,42	Positive
	S6	39,32	34,07	28,97	Positive
	S7	34,62	31,59	28,51	Positive
	S8	33,53	28,29	28,59	Positive
	S9	38,17	35,99	27,78	Positive
	S10	35,53	32,2	27,49	Positive
	S11	–	–	31,79	Negative
	S12	–	–	33,83	Negative
	S13	–	–	30,89	Negative
	S14	–	–	32,83	Negative
	S15	–	–	31,81	Negative
	S16	–	–	29,88	Negative
	S17	–	–	29,52	Negative
	S18	–	–	29,54	Negative
	S19	–	–	32,86	Negative
	S20	–	–	30,13	Negative

Ct: cycle threshold.

The current results indicate that the heat-shock method is reliable for use in the clinical field, even in laboratories lacking centrifugation or cooling equipment. In developing countries like Indonesia, this inexpensive technique can mitigate the cost and supply issues associated with nucleic acid-based testing. In magnetic beads-based automated NAE, varying magnetic rod speed is essential to increase extraction efficiency, while the heating step has negligible effects. These findings can be used as a reference for developing local NAE machines in developing countries.

The magnetic beads-based NAE automatic device is used for diagnostic purposes in hospitals, clinics, forensic laboratories, and pharmaceutical companies. However, these devices are produced by a limited number of countries, such as Canada, Germany, the USA, Singapore, and China. This can lead to testing limitations in a pandemic, particularly for non-device-producing countries such as Indonesia, which delays patient treatment. However, such situations encourage governments to promote the production of local reagents and instruments to improve their responses to future health emergencies [5]. Therefore, NAE optimization methods can overcome the bottlenecks associated with large sample testing, particularly during a pandemic or for high-prevalence diseases that require rapid diagnostic processes.

CRediT author statement

Hesti Lina Wiraswati: Conceptualization, Formal analysis, Funding acquisition, Investigation, Project administration, Supervision, Writing - Original draft preparation, Reviewing and Editing; Ilma Fauziah Ma'ruf: Formal analysis, Visualization, Writing - Original draft preparation, Reviewing and Editing; Savira Ekawardhani: Data curation, Project administration, Validation; Lia Faridah: Data curation, Project administration, Validation; Amila Lelalugina: Data Curation, Investigation, Project administration; Harry Septanto: Methodology, Resources, Software; Imam Damar Djati: Methodology, Resources, Software; Shabarni Gaffar: Formal analysis, Methodology, Supervision; Asif Awaludin: Conceptualization, Funding acquisition, Software, Supervision, Writing - Original draft preparation.

Declaration of competing interest

The authors declare that there are no conflicts of interest.

Appendix A. Supplementary data

The following is the Supplementary data to this article.