Portable and simultaneous detection of four respiratory pathogens through a microfluidic LAMP and real-time fluorescence assay†

Junwen Liu^a, Zhi Zeng^b, Feina Li^c, Bo Jiang^a, You Nie^a, Guohao Zhang^d, Biao Pang^d, Lin Sun*^c and Rongzhang Hao*^a
aSchool of Public Health, Capital Medical University, Beijing 100069, China. E-mail: hao@ccmu.edu.cn
^bSchool of Pharmacy, China Medical University, Shenyang 110122, China
^cBeijing Key Laboratory of Pediatric Respiratory Infection Diseases, Key Laboratory of Major Diseases in Children, Ministry of Education, National Clinical Research Center for Respiratory Diseases, Laboratory of Respiratory Diseases, Beijing Pediatric Research Institute, Beijing Children's Hospital, Capital Medical University, National Center for Children's Health, Beijing 100045, China. E-mail: sunlinbch@163.com
^dBeijing Baicare Biotechnology Co., Ltd, Beijing 102206, China

First published on 4th September 2024

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1. Introduction

In the post-COVID-19 era, the outbreaks of respiratory tract infectious diseases result in increased patient mortality and healthcare costs.^1–4 Respiratory tract infections are common in children⁵ with acute onset and rapid progress, which cause huge social and economic problems with significant implications for children, family and the health care system.^6,7 Most respiratory infection diseases have multiple causative pathogens, such as SARS-CoV-2, human respiratory syncytial virus type A (RSVA), Mycoplasma pneumoniae (MP) and influenza A(H1N1),⁸ but it is difficult to confirm specific pathogens only based on symptoms.⁹ Notably, co-infections occur frequently and can lead to increased disease severity and complications,^10,11 which adversely impact childhood quality of life. Therefore, the simultaneous detection of multiple pathogens in a single assay is urgent for timely diagnosis and accurate treatment, especially in resource-limited regions.

A molecular biology method, known as the polymerase chain reaction (PCR), is considered the gold standard for the detection of infectious diseases with high sensitivity.^12,13 However, the PCR relies on central laboratories, specialized operators and expensive thermal cycling instruments, making it unsuitable for application at the site of an infectious disease outbreak or in resource-poor areas.^14–16 In contrast, isothermal amplification methods can complete nucleic acid amplification at a constant temperature in a short time.¹⁷ Various methods have been developed including loop-mediated isothermal amplification (LAMP),¹⁸ recombinase polymerase amplification (RPA),¹⁹ rolling circle amplification (RCA),²⁰ and strand displacement amplification (SDA).²¹ Among these methods, LAMP is preferred owing to its multiple advantages. It runs under a single temperature condition, feasibly set at 60–65 °C, requiring only one enzyme.²² Owing to the use of multiple primers, LAMP shows increased specificity.^23,24

Multi-channel microfluidic chips have been developed as novel detection platforms in recent years, which are easy to operate at the site and realize multiplexed assays.^25,26 They are more suitable for realizing highly sensitive and rapid testing in resource-poor areas.^27–30 However, multi-channel microfluidic chips still suffer from the shortcomings of expensive equipment costs or difficulties in stable control.

The platforms of centrifugal microfluidic chips have been established by several researchers. The instrument can automatically carry out the processes of sample transfer, data acquisition and result output, therefore simplifying the manual operation process and reducing the variability of the results.^31–33 The combination of centrifugal microfluidics and isothermal amplification provides accurate and rapid screening results in a short time through fluorescence detection.³⁴ However, centrifugal microfluidic chips require complex multilayer structural designs, and the need for equipment that can perform centrifugal operation, simultaneous module heating, data acquisition and signal output increases the size and cost of the equipment. Finger-driven chips do not require complex instrumentation, and liquid flow and mixing between reaction cells can be realized only by finger pressure, which reduces the complexity of operation.^35,36 The operator needs to control the force and direction of the pressure to ensure smooth and accurate liquid flow, and the liquid stored in the blisters flows erratically and may cause an overflow of the preservation cell prematurely due to a pressure error. The combination of lateral flow chromatography and microfluidics can realize a low-cost assay with simple operation, and the expansion to multi-channels can greatly improve the assay efficiency. But it needs an extra-large amount of buffer for the amplification solution of the lateral flow assay, and the chip needs to be turned over to control the liquid flow during the process.^37,38

Thus, it is urgent to develop a low-cost, operationally stable yet simple, sensitive and accurate testing technique for on-site screening of multiple respiratory pathogens in resource-poor areas. The combination of the LAMP assay and the microfluidic chip, which provides a closed environment and simplifies operation, is more conducive to the detection of single infection or co-infection of multiple respiratory pathogens.

Herein, we developed a microfluidic LAMP and real-time fluorescence assay, which contains ten cuvettes with LAMP primers pre-embedded to detect four leading pathogens H1N1, SARS-CoV-2, MP, and RSVA. The sensitivity, specificity and feasibility of the multi-channel LAMP-based microfluidic detection chip were evaluated using commercially synthesized plasmids and clinical specimens. Our findings indicate that the developed method significantly improved the efficiency of on-site detection of respiratory infectious diseases and is a promising diagnostic tool.

2. Materials and methods

2.1 Materials and instruments

All materials were obtained from commercial sources and can be used without further purification. 2× RT-LAMP-Eazy (SYBR plus) was purchased from Jin Yizhen (Suzhou, China), 2× RT-LAMP kit with dye was purchased from Nearshore Protein (Suzhou, China), and DNase/RNase-free deionized water was purchased from Tiangen (Beijing, China). A microfluidic chip, a pressure-sensitive adhesive and a chip cartridge were purchased from Baicare (Beijing, China). Magnetic bead method viral genome extraction kit was purchased from Beaver (Guangzhou, China). A LightCycler® 96 fluorescence quantitative PCR instrument was purchased from Roche (Basel, Switzerland). Oligonucleotides used as LAMP primers and plasmids containing pathogenic sequences to be tested in this study were provided by Sangon Biotech (Shanghai, China).

The microfluidic chip-based LAMP and real-time fluorescence assay was developed to realize portable detection. During the heating process, the cooling sheet served as a temperature adjustment element, while the temperature sensor attached to the heat conduction plate acted as a control feedback element to realize proportional integral derivative (PID) control of the temperature, so as to achieve the purpose of constant temperature control. In this manner, the temperature fluctuation range during the reaction could be restricted to 65 ± 0.5 °C. We employed a single LED lamp bead with a central wavelength of 470 nm as the excitation light source and attached with a band-pass filter and a light guide rod with a central wavelength of 470 nm to ensure that the light intensity deviation did not exceed 5% within the range of 60 × 60 mm² detection bit. The instrument utilized a CMOS camera as the detection element and took cyclic photos of the detection chip to record the reaction changes during the detection process.

The pictures captured by the CMOS camera were stored in the computer within the instrument, and the images were recognized through its analysis software. The position of each reaction cell was determined by the marker pin on the chip, and the fluorescence signal intensity of the immediate reaction was acquired by extracting the signal at the center of each reaction cell, thereby transferring the signal intensity value at each time point throughout the entire reaction process into the real-time LAMP signal curve.

2.2 Sample preparation

Plasmids containing target gene segments of the pathogen were used as DNA templates for influenza A(H1N1), SARS-CoV-2, MP and RSVA, and used for primer screening and system evaluation. The sequences of the target genes are shown in Table S1.†

Respiratory samples were collected from Beijing Children's Hospital, Capital Medical University. Nucleic acids were extracted from nasopharyngeal swabs using the magnetic bead method through small portable equipment such as magnetic racks and metal baths. The DNA of MP and the RNA of the three viruses including RSVA, H1N1 and SARS-CoV-2 were extracted separately.

2.3 Design and screening of LAMP primers

LAMP primers for RSVA and MP were designed using PrimerExplorer V5 online software (https://primerexplorer.jp/lampv5/index.html) and synthesized by Sangon Biotech (Shanghai, China) (Tables S1 and S2†). Five primers of RSVA and four primers of MP were selected for optimal detection. The primers for H1N1 and SARS-CoV-2 were obtained from previously reported studies.^30,39 Primer concentrations were as the following: 1.6 μM of each inner primer (FIP and BIP), 0.2 μM of each outer primer (F3 and B3), and 0.8 μM of each loop primer (LF and LB).

A 25 μL reaction mixture comprised the following: 12.5 μL of 2× RT-LAMP-Eazy (SYBR plus) premix, 5 μL of 5× primer mix, 2.5 μL of DNase/RNase-free deionized water, and 5 μL of template nucleic acid. First, the inner and outer primers of each pathogen were screened using plasmids at a concentration of 10⁸ copies per mL. Amplification curves were obtained using a LightCycler® 96 fluorescence quantitative PCR instrument and primers with an earlier peak onset, good reproducibility and smooth baseline were selected. The corresponding loop primers were then screened and the primer set with the best detection effect contained 4–6 LAMP primers. The fluorescence detection diagram of primer screening is shown in Fig. S1 and S2,† and the best candidate primer sequences chosen are listed in Table 1.

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2.4 Sensitivity and specificity of the LAMP detection system

To evaluate the sensitivity of the established LAMP system, serially diluted plasmids containing a pathogen sequence from 10⁸ copies per mL to 10² copies per mL in a gradient (dilution ratios of 1, 1/10, 1/100, 1/1000, 1/10000, 1/100000 and 1/1000000) were prepared. The DNA plasmid was used as the target in the preliminary stage for RNA viruses, which need to reverse transcribe the RNA to DNA for further LAMP amplification.

For specificity testing, the cross-amplification of the LAMP primers was evaluated. Plasmids from a panel of pathogens at a concentration of 10⁸ copies per mL were added to an assay unit containing each specific LAMP primer of pathogens, and the fluorescence profile generated by the amplification reaction was used to determine whether non-specific amplification occurs.

2.5 Microfluidic chip assembly

The microfluidic chip was fabricated from a polycarbonate (PC) material with a length of 75 mm and a width of 28 mm. The LAMP chamber is located in the middle cavity of the chip with a width of 1 mm, a length of 2 mm and a depth of 2 mm with a volume of about 4 μL. The microfluidic chip comprises a cover sheet and a bottom sheet. The cover and the bottom sheet were closely combined to ensure tightness.

Then, 1 μL of the LAMP primer mixture, i.e., target primers, positive controls (containing a matched nucleic acid template and primers) and blank controls (without primers), was loaded in ten different microchambers of the microfluidic chip and dried in a clean air environment at room temperature. Air-impermeable membranes were covered on the microfluidic channels. Each pathogen had two parallel microchambers to increase the accuracy of the results (1: positive control, 2: MP primer, 3: MP primer, 4: H1N1 primer, 5: H1N1 primer, 6: RSV-A primer, 7: RSV-A primer, 8: SARS-CoV-2 primer, 9: SARS-CoV-2, and 10: blank control). The final concentrations of the primers were the same as those in the LAMP reaction in section 2.3. The microfluidic chip was assembled with the chip cassette as a single unit (Fig. 1) and stored at 4 °C or room temperature for the following detections.

2.6 Evaluation of the microfluidic LAMP and real-time fluorescence assay

The volume of the entire reaction system was 75 μL, including 37.5 μL of 2× RT-LAMP-Eazy (SYBR plus) premix, 22.5 μL of DNase/RNase-free deionized water, and 15 μL of DNA template. The mixed reaction solution was injected gently into individual microchambers through the microchip injection wells to avoid the generation of air bubbles which may affect the acquisition of fluorescence data. The injection ports and channels were sealed with a card box, and then the microchips were inserted into the corresponding detection holes of the detection instrument. The isothermal amplification reaction was set at 65 °C for 60 minutes to obtain an amplification curve. To facilitate the comparison between various microfluidic LAMP chambers, the RFU (relative fluorescence unit) was obtained through dividing every fluorescence value at each time point by the value at the beginning of LAMP reaction. The peak time of LAMP in the tube or microfluidic chip was also obtained when the fluorescence reached 1.3 times its original value to compare the speed and the sensitivity of the assays.

2.7 Evaluation of the microfluidic LAMP and real-time fluorescence assay with clinical samples

The clinical samples, 39 pharyngeal swabs, were collected from Beijing Children's Hospital, Capital Medical University. Nucleic acids were extracted by the magnetic bead method, and then tested by both the fluorescence quantitative PCR and microfluidic LAMP system. The nucleic acid extraction step involved lysing the virus with buffer containing guanidinium isothiocyanate, adsorbing the nucleic acid using magnetic beads, washing the beads in a metal bath and removing impurities as a means of obtaining a higher purity nucleic acid specimen, and the whole extraction procedure took about 40 min. The detection flow of the microfluidic chip is shown in Fig. 2. The fluorescence detection procedure took approximately 60 min to complete. The results were analyzed using the MedCalc software “diagnostic test evaluation calculator” to evaluate the specificity and sensitivity, as well as the 95% confidence interval (https://www.medcalc.org/calc/diagnostic_test.php).

3. Results and discussion

3.1 Sensitivity and specificity of the selected primers

The sensitivity and specificity of the four pairs of LAMP primers and the reaction system were measured in tubes to verify their performance. The plasmids containing the influenza A(H1N1), SARS-CoV-2, MP, and RSVA sequences were tested as assay targets and a series of 10-fold diluted quantitative plasmids were used as templates to determine the sensitivity of the LAMP assay. As shown in Fig. 3A, the DNA amplification products gradually increase, the SYBR Green dye embedded in the DNA double-strand releases fluorescence. When the detection curve peaks, it indicates the opening of the exponential amplification phase. With high amplification efficiency, the fluorescence value tends to stable, indicating that the exponential amplification phase ends and the detection enters the plateau period. Except for SARS-CoV-2, the other three pathogens can reach the maximum fluorescence value or enter the detection of the plateau period at low concentrations. LAMP primers for H1N1, SARS-CoV-2 and RSVA can detect the target DNA as low as 10³ copies per mL. For MP, it can be detected down to 10⁴ copies per mL. As the nucleic acid template concentration decreases, the peak time of the amplification curve gradually delays or even disappears. For each pathogen, the entire LAMP reaction can be completed within 60 minutes. The control without template (blank) showed no detectable signal, indicating no non-specific amplification. As shown in Fig. S3,† the LAMP peak time in the tube presents good linear relationship along with the logarithm of the concentration of nucleic acid for the four respiratory pathogens.

For the specificity tests of the LAMP primers, templates of different pathogens were used to see if they would cause amplification reactions. The template concentrations for each pathogen were all 10⁸ copies per mL. As shown in Fig. 3B, the LAMP primers for each pathogen could detect the corresponding target and no false-positive results were generated, suggesting that the primers have strong specificity. Taking RSVA as an example, it could only detect the template of RSVA and produce the corresponding fluorescence signals, while for the templates of other pathogens, the primers cannot recognize and amplify the sequences, thus resulting in negative results. Therefore, all four pairs of primers used in this study have good sensitivity and specificity and can meet the detection needs of clinical testing.

3.2 Sensitivity and specificity of microfluidic LAMP detection methods

To validate the detection performance of the microfluidic LAMP and real-time fluorescence assay, sensitivity and specificity of the experiments were evaluated. Enzyme-free water was pre-embedded on the microfluidic chip as a blank control and different pathogen LAMP primers were pre-embedded in the remaining reaction pool, as described in the Experimental section. The preparation of DNA templates was similar to the process of validation of the assay performance of the chip. As shown in Fig. 4A, the LAMP primers for H1N1, SARS-CoV-2 and RSVA could detect the target DNA as low as 10³ copies per mL and MP could detect as low as 10⁴ copies per mL, which are comparable to the detection performance of the off-chip detection system. When the assay was performed on the chip and the fluorescence data were collected in real time by the fluorescence detection instrument accompanying the chip, the fluorescence curve generated was messy compared with that of the PCR instrument, which may be due to the lack of an automatic calibration and calculation program for the fluorescence value of the instrument. However, the amplification curves of each template concentration could still be regarded as a gradient difference and the onset time of the amplification curves was gradually shortened with increasing template concentration. As shown in Fig. S4,† the LAMP peak time for microfluidic LAMP also presents good linear relationship along with the logarithm of the concentration of nucleic acid for the four respiratory pathogens.

The specificity results of the chip are shown in Fig. 4B. Each primer set still produced positive detection signals on the chip only for the corresponding pathogen sequences, and there was no cross-contamination between the chip channels that could lead to false-positive results. Compared with the detection value of fluorescence quantitative PCR, the detection baseline fluorescence value of the microarray detection instrument is higher, thus resulting in a smaller numerical difference between the fluorescence values of positive detection and negative detection, which may be related to the fluorescence module of the instrument. However, there are still differences between the positive curve and negative curve, which do not affect our judgment of the results. Moreover, the specificity of the microfluidic LAMP assay could also be ensured through its peak time for the four respiratory pathogens, as shown in Fig. S5,† while the non-target pathogen assay could not present peak time during the whole detection procedure. In addition to using the four target pathogens, we also tested their response towards 6 other pathogens including adenovirus (AD1), group B streptococcus (GBS), influenza A H5N1 virus (H5N1), influenza A H7N9 virus (H7N9), Haemophilus influenzae B (HIB) and Streptococcus pneumoniae (SPN), and the microfluidic LAMP assay presented good specificity.

All chips are single-use and should be sorted into a sealed bag according to medical waste after use. The results of the sensitivity and specificity tests show that microfluidic chip-based LAMP and real-time fluorescence assay shows good detection performance. At the same time, the chip was sealed during the amplification reaction process to reduce aerosol contamination. The chip is easy to operate, only need to put into the instrument slot and start the program to get the real-time detection curve, even inexperienced people can successfully complete the whole process of operation, which is user-friendly. The multiple channels of the chip make it possible to complete the detection of multiple pathogens at one time, which greatly saves the volume of the samples used, and the detection time of only one hour greatly improves the detection efficiency, which is suitable for on-site testing. However, the current instrument contains four card slots, which can only support the simultaneous detection of four samples, and the detection throughput needs to be improved again to meet the demand for large-volume detection during on-site testing. Moreover, early screening for key patients during the outbreak of respiratory infectious diseases can detect pathogens in time and take targeted measures.

3.3 Clinical sample test

Nucleic acid extraction and the microfluidic LAMP and real-time fluorescence assay were validated on the samples provided by Beijing Children's Hospital, Capital Medical University, and these results were compared with those of the real-time fluorescence quantitative PCR method. The microarray detection system and primers were optimized to achieve better detection results in clinical samples testing. Thirty-nine samples were identified by qPCR. According to the qPCR identification results shown in Fig. 5A, 16 influenza A(H1N1) infections, 8 MP infections, and 1 MP-SARS-Cov-2 co-infection and 14 samples with negative results were identified. Detection thresholds were delineated by the kit instructions, where Ct values for the negative samples exceed of all cycle counts because they did not produce an S-curve. Detection of the microfluidic chip-based LAMP and real-time fluorescence assay is based on the onset time that begins to produce an S-shaped fluorescence profile, which is the time point corresponding to the onset fluorescence value that is statistically different from the negative fluorescence threshold. The full assay time was 60 minutes, but the onset of peak time to produce a valid positive result needs to be less than 55 minutes, as a fluorescence curve that begins to rise after 55 minutes will have difficulty in reaching a fluorescence value at the end of the assay that is statistically different from that of the negative group. According to Fig. 5B, the SARS-CoV-2 microfluidic chip-based LAMP and real-time fluorescence assay showed that among the 39 samples, 14 were infected with influenza A(H1N1), 8 were infected with MP, 1 was co-infected with MP and SARS-CoV-2, and 16 were negative, indicating that the microfluidic LAMP technology could successfully differentiate multiple respiratory pathogens, and samples with co-infections could be detected within the same microchip, which could effectively save the time and cost of testing for a large number of samples with unknown pathogen infections. Statistical analysis was performed using the online “diagnostic test evaluation calculator” and the results are shown in Fig. 5C. From the ROC curves obtained after the statistical analysis shown in Fig. 5D, the sensitivity, specificity and AUC of the microfluidic chip-based LAMP and real-time fluorescence assay was 92.00%, 100.00% and 0.975, respectively. The results proved that the established method has high reliability and accuracy in detecting actual samples. Two influenza A(H1N1) samples were tested negative by the developed assay and positive by qPCR. The reason may be the easy degradation of the RNA samples, which might further reduce the nucleic acid content of the samples due to the long storage time and repeated freezing and thawing, finally resulting in the detection error. More importantly, the microfluidic LAMP and real-time fluorescence assay could detect multiple pathogens at the same time within one hour, which was faster than qPCR technology and suitable for on-site detection of multiple respiratory pathogens.

4. Conclusion

In summary, in order to meet the demand for rapid, sensitive, easy-to-operate and miniaturized detection in resource-poor areas, we developed a microfluidic LAMP and real-time fluorescence assay. It contains ten microfluidic channels for isothermal amplification and the real-time fluorescence detection module of multiple respiratory pathogens. The assay uses a microfluidic chip with multiple channels pre-embedded with LAMP primers for different pathogens to provide a sealed and independent reaction environment and avoid aerosol contamination and false-positive results caused by the high concentration of nucleic acid templates during the amplification process. Only one up-sampling with a pipette is required to fill all reaction pools with reaction solutions and templates, which is simple, easy-to-implement, and user-friendly. The sample volume is 75 μL, which saves the consumption of samples and reagents for multi-pathogen testing. Four chips can be accommodated in a small instrument, enabling simultaneous detection and differentiation of different pathogens in four samples. Nucleic acid extraction can be performed manually using portable magnetic devices such as magnetic racks and small heating devices such as metal baths, which is convenient and fast without the need for additional large nucleic acid extraction instruments. Multi-channels enable simultaneous detection of a wide range of respiratory pathogens including influenza A(H1N1), MP, RAVA and SARS-CoV-2, improving detection efficiency. Detection of each pathogen can be as low as 10⁴ copies per mL or 10³ copies per mL. The results of 39 clinical samples with respiratory infections show that the system can detect respiratory pathogens with a sensitivity of 92.00% and a specificity of 100.00%, which meets the detection requirements of pathogen on-site screening. It can effectively detect the pathogens in the clinical samples and show good consistency with qPCR technology. Moreover, the method is more time-saving than the commercial qPCR. The microfluidic LAMP and real-time fluorescence assay can realize rapid and sensitive detection with easy operation, which has significant advantages in on-site detection. In addition, we can develop more channels of microfluidic chips, integrate LAMP primers for different pathogens, and expand the detection slot of the instrument to realize high-throughput respiratory pathogen screening in this field.

Author contributions

Junwen Liu: methodology, experimental design, data analysis, and writing – original draft preparation. Zhi Zeng: methodology, experimental design, data analysis, and writing – original draft preparation. Feina Li: methodology, experimental design, and writing – original draft preparation. Bo Jiang: methodology and data analysis. You Nie: methodology. Guohao Zhang and Biao Pang: data analysis, writing – reviewing and editing, and supervision. Lin Sun: conceptualization, writing – reviewing and editing, and supervision. Rongzhang Hao: funding acquisition, writing – reviewing and editing, and supervision. All authors reviewed the manuscript.

Data availability

The data supporting this article have been included as part of the ESI.†

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was sponsored by the National Key Research and Development Program of China (No. 2021YFC2301102), the Beijing Nova Program (No. 20230484410), the Capital's Funds for Health Improvement and Research (No. 2022-2G-4263), the Beijing Municipal Natural Science Foundation (No. L234051) and the Training Plan for High-Level Public Health Technical Talents of Beijing Municipal Health Commission (2022-02-04 and 2024-03-18).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4an00748d

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