Abstract
Objective
The real-time PCR system is one of the most powerful research tools available in the life sciences field. The aim of this study was to preliminarily evaluate the analytical performance of QuantStudio 1 Plus real-time PCR system (QS 1 plus) for clinical procedures.
Methods
The consistency of QS 1 plus with the reference system in terms of various clinical procedures was evaluated. For qualitative data, the Kappa test was used to analyze the agreement of the results. For the quantitative data, Passing-Bablok regression analysis and Bland-Altman plot analysis were used to assess the concordance between QS 1 plus and the reference instrument.
Results
Passing-Bablok regression showed an excellent agreement between the QS 1 plus and LC 480 systems for HBV DNA quantification (y = 0.928 + 0.970x), whereas Bland-Altman plot analysis showed very small mean deviations between the two systems. The QS 1 plus yielded perfectly consistent results with the reference instrument for methylenetetrahydrofolate reductase (MTHFR) C677T melting curve genotyping analysis, MTHFR C677T genotyping analysis, Norovirus RNA negative/positive analysis, influenza B virus (Flu B) RNA negative/positive analysis, Mycobacterium tuberculosis (MTB) DNA negative/positive analysis, Human Papillomavirus (HPV) genotyping analysis, epidermal growth factor receptor (EGFR) gene mutation analysis. Both the relative quantitative analysis and the relative quantitative analysis (standard curve) confirmed the satisfactory concordance between the QS 1 plus instrument and the ABI 7500 instrument by Passing-Bablok regression analysis (y = 0.180 + 0.817x and y = 0.012 + 1.000x, respectively) and Bland-Altman plot analysis.
Conclusions
Our research has proven that QS 1 plus is adaptable to most test procedures in the clinical laboratory. This may provide the basis for its further application.
Keywords: Polymerase chain reaction, QS 1 plus, Genotyping, Gene expression analysis
Highlights
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The first Chinese-made instrument in the QuantStudio family was evaluated.
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QS 1 plus is comparable to well-established PCR instrument.
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QS 1 plus is adaptable to most test procedures in the clinical laboratory.
1. Introduction
Since the theory of the double helix structure of DNA was proposed in 1953 [1], the study of nucleic acids has been intensified. The establishment of the polymerase chain reaction (PCR) technique in 1985 [2], which enabled the efficient and rapid amplification of target DNA sequences in vitro, became a pioneering achievement in the life sciences. Currently, PCR technology is playing an irreplaceable role in life sciences, genetics, and medicine, especially in pathogenic diagnosis and the detection of genetic mutations. However, the fact that conventional PCR products necessitate “open-cap” analysis increases the risk of contamination, which limits their further application. Moreover, it is difficult to achieve accurate quantification of target genes by conventional PCR techniques due to several factors (amplification efficiency, plateau effect and detection system). On this basis, real-time PCR, which combines PCR technology and fluorescent reporter chemistry, has become a highly sensitive and specific method for monitoring the process of template DNA amplification [3]. Nowadays, real-time PCR technology is flourishing in the medical field, especially in response to the rapid diagnosis of novel pathogens. As an example, real-time PCR-based detection systems have become the gold standard for COVID-19 diagnosis due to their quantitative, accurate, sensitive, and rapid characteristics [4].
Despite the numerous advantages of PCR technology, a PCR instrument with precise temperature control, multiple fluorescence channels and accurate fluorescence acquisition is also decisive for success or failure. Currently, there are various real-time PCR instruments available on the market for clinical purposes in China, such as Roche LightCycler 480 (LC 480) instrument and Applied Biosystems 7500 (ABI 7500) instrument. The ABI 7500 real-time PCR system can run a range of applications for the laboratory in around 2 h, including gene expression analysis, absolute quantitation, single nucleotide polymorphism (SNP) genotyping, positive/negative assays utilizing internal positive controls, and high-resolution melting (HRM) analysis. Similarly, the LC 480 real-time PCR system offers the advantages of rapidity, high throughput as well as stable fluorescence acquisition, and has been widely used for absolute/relative quantitative, endpoint genotyping, melting curve genotyping, DNA methylation studies, micro-RNA (miRNA) studies, and HRM scanning. Unfortunately, notwithstanding the benefits of the above-mentioned real-time PCR systems, they are prohibitively expensive, have high maintenance costs and the all-English operating system is not user-friendly for application in the community medical institutions in China. Additionally, these instruments have been available for more than a decade, with cumbersome machines and backward software that do not meet today's demands for intelligence, automation, and interactivity. Consequently, there is an urgent need for a real-time PCR system that is economical and suitable for use by most Chinese healthcare institutions.
The Applied Biosystems QuantStudio 1 Plus real-time PCR system (QS 1 plus) is the first Chinese-made instrument in the QuantStudio family. The Chinese language operating interface makes it user-friendly for Chinese users. The system design remains the strength of ABI real-time PCR systems and provides high quality, excellent reliability, and outstanding user experience. The QS 1 Plus real-time PCR system is equipped with an interactive touch screen interface, intuitive software, and pre-optimized program templates, as well as a web browser and computer desktop analysis option. Data can be freely analyzed and shared in conjunction with Thermo Fisher cloud services. The aim of this study was to preliminarily evaluate the analytical performance of QS 1 plus for clinical procedures by comparison with the real-time PCR systems which are now well-established in clinical laboratories.
2. Materials and methods
2.1. Overall design
This study was designed to compare the analytical performance for clinical tests of the QS 1 plus with the ABI 7500 or LC 480, which have been approved by the National Medical Products Administration of China. This research was conducted according to two internal documents (PUMCHL-Mo-2-Q10-05 Performance validation procedures for qualitative experiments in molecular biology projects and PUMCHL-Mo-2-Q11-03 Performance validation procedures for quantitative experiments in molecular biology projects) of our laboratory, which mainly refer to the China National Accreditation Service for Conformity Assessment (CNAS) guidelines [[5], [6], [7]]. We selected the detection systems corresponding to the current testing projects as the references, which are regularly inspected by CNAS ISO 15189 and College of American Pathologists (CAP). Significantly, the ABI 7500 and LC 480 instruments we used were fully evaluated in their performance validation prior to the project being undertaken. Furthermore, the ABI 7500 and LC 480 in our laboratory are regularly calibrated and maintained to ensure their accuracy. The performance of the QS 1 plus was evaluated in two parts, one by a professional organization for the machine performance of the QS 1 Plus itself, and the other by our laboratory for a preliminary assessment of its clinical application value. The QS 1 plus has been assessed by the Jiangsu Institute of Medical Devices Testing for machine performance evaluation according to relevant industry standards and has been endorsed (Project number: 2022QW2339). For all evaluated clinical testing items, control material was assayed along with the samples in each batch. The main types of application include: absolute quantitative analysis (standard curve), melting curve analysis, genotyping analysis, negative/positive determination analysis, relative quantitative analysis, relative quantitative analysis (standard curve) (Fig. 1).
Fig. 1.
The overall design of this study.
2.2. Absolute quantitative analysis (standard curve)
A total of 25 plasma samples were collected. The extracted DNA from plasm was used as the template for the simultaneous assays on the QS 1 plus and LC 480 using the HBV DNA Quantitative Assay Kit (DAAN Technology Co. LTD, Guangdong, China). Four HBV standards of 2 × 103, 2 × 104, 2 × 105, 2 × 106 IU/ml were used to fit the standard curve and to quantify HBV DNA in the patient samples.
2.3. Melting curve genotyping analysis
The whole blood of 20 patients was collected and nucleic acids were extracted by using the Tianlong extraction reagents and matching instruments (Tianlong Technology Co. LTD, Xi’an, China). The C677T gene polymorphism of the methylenetetrahydrofolate reductase (MTHFR) gene was determined by using the Human MTHFR (C677T) Gene Polymorphism Detection Kit (PCR-Melting Curve Method, Ruiqi Technology Co. LTD, Jiangsu, China). The above procedures were completed simultaneously on the QS 1 plus and the ABI 7500 to assess the consistency between the two systems.
2.4. Genotyping analysis
The nucleic acids were extracted from whole blood collected from 20 patients. Genotyping was then performed simultaneously on the QS 1 plus and ABI 7500 using the Human MTHFR (C677T) Gene Polymorphism Detection Kit (PCR-Fluorescent Probe Method, Youzhiyou Technology Co. LTD, Wuhan, China). The FAM, VIC and ROX channels are used to detect the C allele, T allele and internal control (IC) fluorescence, respectively.
2.5. Norovirus RNA negative/positive analysis
Twenty stool samples from patients with diarrhea were collected and then nucleic acid extraction was performed. Nucleic acid extraction of stool samples was carried out using Tianlong extraction reagents and matching instruments (Tianlong Technology Co. LTD, Xi’an, China). PCR amplification was performed on the QS 1 plus and ABI 7500 respectively using the Norovirus Nucleic Acid Detection Kit (PCR-Fluorescent Probe Method, Langde Technology Co. LTD, Hubei, China). The FAM channel detects the presence or absence of norovirus RNA and the VIC channel monitors the amplification process of exogenous IC.
2.6. Influenza B virus (Flu B) RNA negative/positive analysis
Pharyngeal swab samples were collected from 20 patients with suspected Flu B infection and nucleic acids were extracted. The presence or absence of Flu B RNA was determined using the Respiratory Pathogen Nucleic Acid Detection Kit (PCR-Fluorescent Probe Method, Shengxiang Technology Co. LTD, Hunan, China) in both the QS 1 plus and ABI 7500 systems. The amplified fluorescence of Flu B and IC was captured using the FAM and ROX channels, respectively.
2.7. Mycobacterium tuberculosis (MTB) DNA negative/positive analysis
Sputum samples were collected from 20 patients with suspected MTB infection. Nucleic acid extraction was performed on sputum specimens using a TB-specific Nucleic Acid Extraction Kit (Tianlong Technology Co. LTD, Xi’an, China). PCR amplification was done on the QS 1 plus and ABI 7500 respectively according to the instructions of the MTB Nucleic Acid Detection Kit (PCR-Fluorescent Probe Method, Boaojingdian Technology Co. LTD, Beijing, China).
2.8. Human Papillomavirus (HPV) genotyping analysis
Genital tract swabs from 20 patients with confirmed HPV infection were collected and nucleic acids were extracted using a validated Nucleic Acid Extraction Kit (Magnetic Bead Method, Zhijiang Technology Co. LTD, Shanghai, China). The HPV Genotyping Kit (PCR-Fluorescent Probe Method, Zhijiang Technology Co. LTD, Shanghai, China) was used to qualitatively detect specific DNA fragments of HPV high-risk types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 66, 82 as well as low-risk types 6 and 11 in genital tract secretions. Experiments were run simultaneously on both the QS 1 plus and ABI 7500 systems to assess the consistency of the results between them.
2.9. Epidermal growth factor receptor (EGFR) gene mutation analysis
The QIAGEN Nucleic Acid Extraction Kit (QIAGEN Technology Co. LTD, Germany) was used to extract nucleic acid from tumor tissue samples of 24 lung cancer patients. The Human EGFR Mutation Detection Kit (Fluorescent PCR method, Aide Technology Co. LTD, Xiamen, China) was used to detect 7 common mutations in EGFR, including 19-Del, L858R, T790M, 20-Ins, G719X, S768I and L861Q. The FAM and VIC channels are used to monitor the amplification process of EGFR mutations and IC, respectively. The above tests were performed on both the QS 1 plus and the currently established LC 480 system.
2.10. Relative quantitative analysis
Serum samples from 15 patients with hepatitis C were collected and the miRNA was purified using the TaqMan miRNA ABC Purification Kit (Thermo Fisher Scientific, USA) according to the instruction. The cDNA was prepared using the TaqMan Advanced miRNA cDNA Synthesis Kit (Thermo Fisher Scientific, USA), following the manufacturer's instruction. PCR amplification was performed on QS 1 plus and ABI 7500 using the cDNA as template and adding hsa-miR-122 or hsa-miR-16 primer/probe mixtures. Sample #1 was set as the reference sample and hsa-miR-16 as the reference gene.
2.11. Relative quantitative analysis (standard curve)
Serum samples from 12 patients with hepatitis C were collected and cDNA was prepared according to the above steps. Sample #9 was selected as the standard for hsa-miR-122 and sample #1 was selected as the standard for has-miR-16. Four concentration gradient standards were obtained by serial 5-fold dilution of sample #9 and sample #1, and then the relative standard curves were established. PCR amplification was performed on the QS 1 plus and ABI 7500 respectively. Sample #5 was set as the reference sample while hsa-miR-16 as the reference gene and the relative quantification values were subsequently calculated for each sample.
2.12. Statistical analysis
Data management and statistical analysis are performed using Excel 2019 (Microsoft Inc., United States), MedCalc software (version 19.6.1), R Project (version 4.2.0) and RStudio (Open-Source Edition) software. For qualitative data, the Kappa test was used to analyze the agreement of the results between the QS 1 plus and the reference instrument. For the quantitative data, Passing-Bablok regression analysis and Bland-Altman plot analysis were used to assess the concordance between QS 1 plus and the reference instrument. P<0.05 is considered statistically significant.
3. Results
3.1. Absolute quantitative analysis (standard curve)
A total of 25 plasma samples were submitted for HBV DNA testing on the QS 1 plus instrument, while 4 quantitative standards were used to provide quantitative analysis for positive samples. There were 17 samples identified as HBV DNA positive based on the QS 1 plus system, while 8 samples were identified as HBV DNA negative. The LC 480 system also yielded completely consistent results in terms of positive/negative agreement (Kappa = 1, P<0.001). Furthermore, the standard curve was fitted using standards to complete the quantification of HBV DNA in positive samples. The logarithmic values of viral loads obtained on the QS 1 plus and LC 480 systems were subsequently analyzed and compared using Passing-Bablok regression and Bland-Altmam plot analysis (Fig. 2A and 2B). The slope of the Passing-Bablok regression line was 0.970 (95% CI: 0.910 to 1.062) and the intercept was 0.928 (95% CI: 0.688 to 1.806). The Cusum test revealed no significant deviation from linearity (P = 0.23) and the correlation coefficient was 0.994. The Bland-Altmam plot indicated a tiny mean bias (0.87) between the two instruments.
Fig. 2.
HBV DNA quantitative testing on QS 1 plus and LC 480
A, Passing-Bablok regression analysis for the comparison of HBV DNA quantification results on the QS 1 plus and LC 480 systems. B, Bland-Altmam plot analysis for the comparison of HBV DNA quantification results on the QS 1 plus and LC 480 systems.
3.2. Melting curve genotyping analysis
The MTHFR C677T genotype of 20 subjects was determined using melting curve analysis on the QS 1 plus and ABI 7500. The melting peak of the T allele was about 55°C while that of the C allele was about 65°C. QS l plus and ABI 7500 returned consistent identification results: 6 subjects were classified as CC genotype, 8 as CT genotype and the remaining 6 as TT genotype (Table 1, Kappa = 1, P<0.001).
Table 1.
Consistency assessment of various projects on QS 1 plus and ABI 7500.
| Projects | QS 1 plus | ABI 7500 | Kappa | P value |
|---|---|---|---|---|
| MTHFR (C677T) melting curve genotyping analysis (n=20) | ||||
| CC | 6 | 6 | 1 | <0.001 |
| CT | 8 | 8 | ||
| TT | 6 | 6 | ||
| MTHFR (C677T) genotyping analysis (n=20) | ||||
| CC | 6 | 6 | 1 | <0.001 |
| CT | 8 | 8 | ||
| TT | 6 | 6 | ||
| Norovirus RNA analysis (n=20) | ||||
| Positive | 15 | 15 | 1 | <0.001 |
| Negative | 5 | 5 | ||
| Flu B RNA analysis (n=20) | ||||
| Positive | 15 | 15 | 1 | <0.001 |
| Negative | 5 | 5 | ||
| MTB DNA analysis (n=20) | ||||
| Positive | 15 | 15 | 1 | <0.001 |
| Negative | 5 | 5 | ||
| HPV genotyping analysis (n=20) | ||||
| 16 | 1 | 1 | 1 | <0.001 |
| 18 | 2 | 2 | ||
| 31 | 2 | 2 | ||
| 39 | 2 | 2 | ||
| 51 | 1 | 1 | ||
| 52 | 2 | 2 | ||
| 56 | 2 | 2 | ||
| 58 | 2 | 2 | ||
| 59 | 1 | 1 | ||
| 68 | 1 | 1 | ||
| 31,52 | 1 | 1 | ||
| 35,59 | 1 | 1 | ||
| 52,56,66 | 1 | 1 | ||
| 58,59 | 1 | 1 | ||
3.3. Genotyping analysis
The genotyping method was also implemented on the QS 1 plus and ABI 7500 to evaluate the concordance between the two systems in MTHFR genotype identification. The allele type profiles and identification results were presented in Table 1 where it can be found that both systems obtained the same genotyping results for each subject (Kappa = 1, P<0.001).
3.4. Norovirus RNA negative/positive analysis
Twenty fecal samples were determined as negative/positive for norovirus RNA on the QS 1 plus and ABI 7500. Of these, 15 samples were identified as positive for norovirus RNA whilst 5 samples were identified as negative in both systems (Table 1, Kappa = 1, P<0.001).
3.5. Flu B RNA negative/positive analysis
Twenty samples were submitted for the Flu B RNA assay on QS 1 plus and ABI 7500 to compare the consistency of their performance. As illustrated in Table 1, 15 Flu B RNA-positive samples and 5 Flu B RNA-negative samples were confirmed on the QS 1 plus system. The ABI 7500 yielded perfectly consistent identification results with QS 1 plus (Kappa = 1, P<0.001).
3.6. MTB DNA negative/positive analysis
Twenty sputum samples were subjected to MTB DNA negative/positive analysis on the QS 1 plus and ABI 7500 respectively. The results show excellent agreement between QS 1 plus and ABI 7500 in determining negative/positive for MTB DNA (Table 1, Kappa = 1, P<0.001).
3.7. HPV genotyping analysis
HPV genotyping analysis of 20 samples was achieved on the QS 1 plus and ABI 7500 respectively. The QS 1 plus system indicated that 16 patients were infected with single HPV types while 4 patients were infected with mixed HPV types (Table 1). Similarly, fully equivalent results were obtained with the ABI 7500 system (Kappa = 1, P<0.001).
3.8. EGFR gene mutation analysis
Twenty-four tumor tissues were used for EGFR mutation gene testing performed simultaneously on the QS 1 plus and LC 480 instruments. According to the QS 1 plus system, 7 of the 24 patients were identified with 19-Del, 7 with L858R, 6 with other mutations whilst 4 patients had no EGFR mutations (Table 2). The LC 480 system gave identical results to the QS 1 plus (Kappa = 1, P<0.001).
Table 2.
Consistency assessment of EGFR gene mutation analysis on QS 1 plus and LC 480.
| EGFR mutations | QS 1 plus | LC 480 | Kappa | P value |
|---|---|---|---|---|
| 19-Del | 7 | 7 | 1 | <0.001 |
| L858R | 7 | 7 | ||
| L858R + T790 M | 2 | 2 | ||
| 20-Ins | 1 | 1 | ||
| G719X | 1 | 1 | ||
| L861Q | 1 | 1 | ||
| S768I | 1 | 1 | ||
| No mutations | 4 | 4 |
3.9. Relative quantitative analysis
Serum from 18 HCV-infected patients was collected and analyzed to evaluate the relative expression of hsa-miR-122 using sample #1 as a control. The logarithmic values of the relative quantitative values obtained were used to perform Passing-Bablok regression and Bland-Altmam plot to assess the comparability between the two systems. The slope of the Passing-Bablok regression line was 0.817 (95% CI: 0.724 to 0.950) and the intercept was 0.180 (95% CI: 0.000 to 0.284). The Cusum test revealed no significant deviation from linearity (P = 0.17) and the correlation coefficient was 0.978 (Fig. 3A). The Bland-Altmam plot indicated a slight mean bias (0.03) between the two instruments (Fig. 3B).
Fig. 3.
The comparison of has-miR-122 results on the QS 1 plus and ABI 7500 systems
A, Passing-Bablok regression for relative quantification analysis. B, Bland-Altmam plot for relative quantification analysis. C, Passing-Bablok regression for relative quantification analysis (standard curve). D, Bland-Altmam plot for relative quantification analysis (standard curve).
3.10. Relative quantitative analysis (standard curve)
Serial dilutions of sample #9 were used as hsa-miR-122 standards to draw a standard curve for the quantitative analysis for 12 serum samples. The logarithmic values of the relative quantitative values obtained were used to perform Passing-Bablok regression and Bland-Altmam plot to assess the comparability between the two systems. The slope of the Passing-Bablok regression line was 1.000 (95% CI: 0.898 to 1.080) and the intercept was 0.012 (95% CI: -0.049 to 0.101). The Cusum test revealed no significant deviation from linearity (P = 0.86) and the correlation coefficient was 0.995 (Fig. 3C). The Bland-Altmam plot indicated a slight mean bias (0.01) between the two instruments (Fig. 3D).
4. Discussion
Real-time PCR instruments are now widely used for gene expression analysis, genotyping analysis, pathogen detection and gene mutation analysis. As equipment intelligence progresses, there is a growing demand for intelligent, convenient, and localized real-time PCR instruments. The QS 1 plus real-time PCR instrument is a cost-effective Chinese instrument that can be adapted to a wide range of fluorescent dyes (FAM/SYBR Green, VIC, JUN/ROX, Cy5, SYTO9, HEX/TET/JOE, Texas Red). It is worth noting that the QS 1 plus can be offered with the Chinese operating interface and the web browser-based cloud software. These may be of great convenience to the staff of the laboratory department. However, a sound real-time PCR instrument must have superior performance to deliver accurate information for patient diagnosis, treatment, and prognosis. This study used the clinically well-established ABI 7500 or LC 480 systems as references and revealed that the QS 1 plus was superbly comparable to them for the common clinical procedures. To the best of our knowledge, this study is the first report to evaluate the performance of QS 1 plus. These results offered evidence for the clinical application of the QS 1 plus system.
HBV infection, as a global public health issue, can lead to chronic hepatitis and a range of long-term complications [8]. The assessment and management of patients with HBV has gradually shifted from serological to molecular diagnostic tests. Remarkably, quantitative PCR analysis enables precise monitoring of the dose-response response of patients with HBV and effect of different antiviral treatment compounds [9]. In this study, both the QS 1 plus and LC 480 systems identified 17 HBV-positive samples and 8 HBV-negative samples without disagreement. Furthermore, the quantitative values of the positive samples and their logarithmic values were used for analysis and comparison to estimate the bias within the two systems. Passing-Bablok regression is characterized by the fact that specific assumptions about the sample distribution and measurement errors are not required, and systematic and proportional biases of both methods can be specified [10]. Meanwhile, Bland-Altman plots have been widely implemented to assess the agreement of two instruments or two methods [[11], [12], [13]]. The Passing-Bablok regression and Bland-Altman plot confirmed the excellent agreement between QS 1 plus and LC 480 in the quantification of HBV DNA.
With the expansion of genomics, variants of DNA sequences on the human genome, called SNPs, have received increasing attention and are being utilized in clinical investigations [14,15]. However, precise detection of individual base mutation/substitution/insertion/deletion places heavy demands on the detection platform especially the PCR instrument. In this study, we examined the performance of QS 1 plus in genotyping using the MTHFR C677T genotype as an example. The MTHFR C677T mutation can cause a codon change from valine to alanine at position 222, which reduces MTHFR activity, increases homocysteine accumulation and ultimately increases the risk of cardiovascular disease [16]. Melting curve genotyping and sequence-specific PCR are common methods for determining MTHFR genotype in clinical practice. Based on the theory that two allele-specific amplicons of different sizes result in distinct melting temperatures, the melting curve analysis can distinguish different alleles and thus determine the genotype of the subject [17]. In this scenario, the PCR instrument requires continuous monitoring of variations in fluorescence to enable smooth recognition of melting peaks. We found that the melting peak of the T allele was around 55°C whereas the melting peak of the C allele was around 65°C, which was applicable on both QS 1 plus and ABI 7500. Additionally, the QS 1 plus and ABI 7500 yielded perfectly matched results for both melting curve genotyping and specific sequence PCR genotyping. These results strongly support the prospective utility of QS 1 plus for genotyping.
Real-time fluorescence-based quantitative PCR method for negative/positive analysis exhibits remarkable sensitivity and specificity which is widely adopted as a laboratory diagnostic route in clinical settings. To fully understand the capability of QS 1 plus, we selected some common clinical projects to compare its consistency with the established system. Norovirus is an important pathogen that causes acute gastroenteritis in humans. Prompt and accurate identification of norovirus would help to prevent the spread of outbreaks. Real-time PCR-based platform is an accurate and efficient tool for detecting Norovirus at present [18]. Moreover, the high level of impurities and interfering substances in stool poses a challenge for detecting norovirus from it. Influenza B is an important respiratory pathogen as well as a substantial contributor to the morbidity and mortality associated with influenza epidemics [19]. The real-time fluorescent quantitative PCR technique has become an essential reference for the screening of Influenza B. As the pathogen of tuberculosis, MTB is of widespread clinical concern [20]. In terms of detection methods, microscopy and culture methods have some drawbacks (low sensitivity and long turnaround time), while real-time PCR technique targeting the MTB genome is a good choice [21]. Thus, we selected above three common pathogens from various sample types in the clinical setting to evaluate the performance of QS 1 plus. The results indicated the outstanding capability of QS 1 plus as a PCR platform in the identification of infectious pathogens.
HPV infection has been shown to be strongly associated with the risk of cervical cancer and precancerous lesions, and HPV-based screening is broadly recommended for women [22]. In our laboratory, we routinely test for 9 high-risk types (HPV16, 18, 39, 45, 56, 59, 66, 68 and 82), 6 intermediate-risk types (HPV31, 33, 35, 51, 52, and 58) and 2 low-risk types (HPV6 and 11) at the same time. In this context, the four channels (FAM, VIC, TEXAS RED and CY5) need to acquire fluorescent signals simultaneously to suit the detection of these targets. Our study demonstrated that QS 1 plus can accurately identify distinct HPV types despite confronting the complexities of so many targets and multiple fluorescent assays. As critical target in drug concomitant diagnosis, EGFR mutation has gained growing recognition for its prognostic and predictive value in non-small cell lung cancer [23]. Given the scarcity and intricacy of tumor tissue, extracting nucleic acids from it for real-time PCR to obtain information on EGFR mutations places considerable demands on PCR equipment. Fortunately, the QS 1 plus presented satisfactory results in the comparison study. Overall, the QS 1 plus is perfectly consistent with the current well-established system and is expected to be further used in clinical laboratories in China.
As a non-negligible field of real-time PCR technique, relative quantification is used in the areas of genomics and functional transcriptomics for the gene expression analysis in biological experiments. Here we use hsa-miR-122 as an example to judge the effectiveness of QS 1 plus in relative expression analysis and relative expression analysis (standard curve), respectively. It has been reported that has-miR-122 is a unique molecule with enormous potential in the diagnosis, prognosis and treatment of liver diseases [24]. Owing to the short length of mature miRNAs and the high degree of similarity among miRNA family members, there are some specific challenges in terms of expression analysis of non-coding RNA by Real-time PCR [25]. Through Passing-Bablok regression and Bland-Altman plot, the satisfactory consistency between the QS 1 plus and ABI 7500 systems was observed. This endorsed the usefulness of the QS 1 plus in the field of relative quantification.
Inevitably, there were some limitations to this study. This study only evaluated the consistency between QS 1 plus and well-established PCR systems. Some specific aspects, such as the limit of detection for the quantitative results and cut-off analyses for qualitative, were missing. Consequently, this study should be considered as a pilot study to evaluate the performance of the QS 1 plus. Comprehensive performance validation is required before a laboratory would consider running clinical projects on the QS 1 plus or moving existing clinical projects from another PCR system to the QS 1 plus. Our laboratory recommends the assessment of reproducibility, method compliance, lower limit of detection, anti-interference capacity, and cross-reactivity for qualitative projects while evaluations for precision, linearity interval, accuracy, lower limit of detection, cross-reactivity, and anti-interference ability for quantitative projects. In practice, laboratories can establish appropriate validation procedures according to their own conditions and the requirements of some accreditation organizations such as CNAS or College of American Pathologists (CAP).
5. Conclusions
Overall, this study compares the QS 1 plus with current well-established PCR instruments from different perspectives such as absolute quantification, melting curve genotyping, genotyping analysis, negative/positive analysis, and relative quantification. The results of the study proved that the QS 1 plus was perfectly adapted to current clinical testing programs. This study may provide the basis for its further application.
Ethics approval
This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Peking Union Medical College Hospital (No.002912).
Author contributions
YC, YX and ZW contributed to conception and design; JY and RZ collected samples; QY and YL performed the experiments; WY and DZ helped for the data statistical analysis; ZW and YC performed the data statistical analysis and wrote the manuscript. All authors contributed to manuscript revision, and read and approved the submitted version.
Funding
This work was supported by the Beijing Key Clinical Specialty for Laboratory Medicine Excellent Project (No. ZK201000).
Declaration of competing interest
The authors report no competing interests.
Data availability
Data will be made available on request.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
Data will be made available on request.