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BY 4.0 license Open Access Published by De Gruyter December 23, 2022

RNA-based isothermal amplification technology and its clinical application in pathogen infection

  • Jie Teng , Fang Liu , Li Chang , Qiuxia Yang , Guanglu Che , Shuyu Lai , Yuan Tan , Jiaxin Duan and Yongmei Jiang EMAIL logo

Abstract

It is very important to detect pathogenic bacteria, viruses, or fungi in a patient’s secretion or body fluid samples as soon as possible to determine the patient’s recovery. For certain pathogens, the amount of ribosomal RNA copies contained is often tens of thousands of times higher than the amount of DNA copies, so the detection of RNA has higher sensitivity. In addition, whether for DNA pathogens or RNA pathogens, the direct detection of ribonucleic acid transcribed by pathogens in vivo can distinguish active infection or past infection, can eliminate the influence of residual DNA of pathogens that have died in the lesions, and can also avoid excessive medical interventions for transient infections, which is of great significance in the field of infectious pathogen detection. Isothermal amplification technology played important roles in molecular diagnosis because of its significant advantages. Highly sensitive RNA detection can be achieved by both direct transcription amplification and indirect amplification based on reverse transcription. Direct transcription amplification technologies relies on reverse transcriptase and T7 RNA polymerase to achieve linear transcription amplification of RNA on one-step; while the indirect amplification technology depends on a reverse transcriptional process at the beginning of the reaction. Both methods have outstanding advantages in clinical application, and commercial kits and commercial all-in-one machines based on these principles have been put into clinical use. This review mainly introduces the clinical application of isothermal amplification technologies in the detection of RNA pathogens and the main difficulties faced at this stage. It is hoped to provide insightful ideas for the construction of pathogen RNA detection technology to meet the needs of point-of-care testing in the future.

1 Introduction

With the development of molecular biology technology, the detection for pathogen infection no longer relies on microscopic examination or culture method based on seeing “whole pathogen” and immunological method based on detecting “protein component of pathogen” [1,2]. The limitations of low positive rate, long cycle, and low accuracy make it unable to meet the actual needs of clinical precision medicine. Nucleic acid detection technology based on molecular genetic level has become the mainstream development trend of infectious pathogen detection due to its rapid and accurate advantages [3]. It gets rid of the dependence of traditional detection methods on isolation and culture, and significantly improve the sensitivity and specificity by amplifying and detecting specific nucleic acid sequences of target pathogens.

The genetic central dogma proposed by Crick in 1958 pointed out that deoxyribonucleic acid (DNA) is the carrier for storing genetic information, and ribonucleic acid (RNA) is the carrier for transmitting genetic information. According to the genetic material carried by different pathogens, molecular biology technology include the detection of DNA and RNA [4]. Pathogens which use DNA as the main genetic material can be divided into two types: double-stranded DNA pathogens and single-stranded DNA pathogens. While the RNA-containing pathogens can exist only as a single strand. When the body is infected with a certain DNA pathogen and accompanied for a long time, it will transcribe to form mRNA, which then integrate with host cells and translate into protein. If one is infected by a certain RNA pathogen, it is equivalent to obtaining the RNA information, which can be translated directly by the host cells [5]. It is not difficult to understand that the DNA-containing pathogens are typically milder than RNA-pathogens and mutagenize to a lesser degree.

For a pathogen, it may contain only one or a few copies of DNA, but up to 10,000 copies of ribosomal RNA (rRNA) molecules, so the detection of rRNA can greatly improve the sampling efficiency and detection sensitivity. Furthermore, the detection of RNA has greater clinical guiding significance. When the pathogen is dead and the infection is effectively controlled, pathogen RNA information degrades so easily that it is completely undetectable for 2–3 days after cure. Since there are still dead bacteria remaining after the cure, it takes at least 3–4 weeks for the body to completely metabolize the DNA information of the pathogen. Therefore, the detection of RNA is equivalent to culture results, which can effectively distinguish active infection or past infection, can eliminate the influence of residual DNA of pathogens that have died in the lesions after treatment on the test results, and can also avoid excessive medical interventions for transient infections [6,7]. We have to admit that, RNA-based molecular biology technologies are widely used in pathogen detection and showed outstanding advantages.

Nucleic acid isothermal amplification is a new type of nucleic acid amplification technology developed in recent decades [4,8]. It has the advantages of rapid, sensitive, and specific, which can be performed under simple conditions, such as in a thermostatic water bath. And it does not require professional equipment and technical personnel. Therefore, it has huge development advantages in the field of clinical application, especially in resource-poor settings and on-site detection.

This review introduces the isothermal amplification technologies and clinical application in detecting RNA of the pathogen. We hope that it will soon make researchers understand the current research status and application scenarios of RNA-based detection technology and provide insights into the construction of new RNA-based technologies. Finally, challenges and perspectives in the field will be discussed.

2 Isothermal amplification technologies in detecting RNA

2.1 Transcriptional amplification strategy: From RNA to RNA

As the fourth generation of diagnostic technology, RNA molecular diagnostic technology has been widely used in blood screening, infectious disease detection, genetic disease diagnosis, and other fields. The researches on RNA detection strategies relying on transcription amplification technology are becoming more and more popular, because these strategies are more direct, and they can achieve high-efficiency amplification of RNA targeting RNA. Furthermore, RNA is unstable and mostly exists in single-stranded form in nature. The amplified RNA products are extremely easy to degrade, so it does not cause laboratory contamination and the false positive rate of detection is low [9,10]. Transcription-based RNA detection strategies mainly include nuclear acid sequence-based amplification (NASBA) technology of BioMerieux (France), transcription-mediated amplification (TMA) technology of Gen-Probe (America), and simultaneous amplification and testing (SAT) technology of Rendu (China).

2.1.1 NASBA technology

NASBA technology is one of the most widely used isothermal amplification technology in RNA detection [11,12]. It require two primers and three key enzymes, namely, Avian myeloblastosis virus reverse transcriptase (AMV RT), T7 RNA polymerase, and RNA hydrolase (RNase H). The schematic diagram is shown in Figure 1. It is worth mentioning that the whole reaction process is carried out under a constant temperature (∼41℃) without special equipment for precise temperature control, which has been widely used in pathogen detection, environmental microbial detection, and other fields, showing broad application prospects.

Figure 1 
                     The schematic diagram of NASBA reaction: the designed primer 1, containing a T7-promoter sequence, could hybridize with target RNA to initiate primer extension and form RNA–cDNA hybrid chain. RNA was hydrolyzed from the RNA–cDNA hybrid chain by RNase H and formed a single strand of cDNA. The cDNA triggered primer 2 extends and forms a double-stranded DNA intermediate. These intermediates containing T7 RNA polymerase recognition sites were used as templates for in vitro transcription amplification and generated single-stranded RNA products. The products are the complementary strand of the target RNA, so it combined with primer 2 again to enter the next amplification cycle and accumulate a large number of free RNA products.
Figure 1

The schematic diagram of NASBA reaction: the designed primer 1, containing a T7-promoter sequence, could hybridize with target RNA to initiate primer extension and form RNA–cDNA hybrid chain. RNA was hydrolyzed from the RNA–cDNA hybrid chain by RNase H and formed a single strand of cDNA. The cDNA triggered primer 2 extends and forms a double-stranded DNA intermediate. These intermediates containing T7 RNA polymerase recognition sites were used as templates for in vitro transcription amplification and generated single-stranded RNA products. The products are the complementary strand of the target RNA, so it combined with primer 2 again to enter the next amplification cycle and accumulate a large number of free RNA products.

2.1.2 TMA technology

TMA technology is improved and developed based on NASBA [13]. It is suitable for any type of nucleic acid template and can proceed in a single tube. Studies have found that Moloney mouse leukemia virus reverse transcriptase (MMLV-RT) has certain RNase H activity, so only two enzymes (MMLV-RT and T7 RNA polymerase) are needed in this technology to achieve linear transcription amplification of RNA [14].

2.1.3 SAT technology

SAT strategy is a patented technology independently developed by domestic enterprises which combine TMA amplification and real-time signal amplification. Based on TMA technology, it introduces molecular beacon (MB) probe to detect the fluorescence signal in the amplification system in real time. The specific principle is shown in Figure 2.

Figure 2 
                     Schematic diagram represents SAT technology. MBs are probe molecules that exhibit a characteristic stem-loop structure through which the 5′ and 3′ ends are maintained in close proximity. At this time, the fluorescence signals are quenched because the reporter groups and quencher groups are close to each other. The disruption of base-pairing in the stem, upon denaturation, allows the probe to hybridize to a complementary target. In this case, the reporter groups and the quencher groups are separated sufficiently, inducing the reporter groups to restore the fluorescence signal. Thus, the target can be determined based on the real-time increase in the fluorescence signal in the reaction system.
Figure 2

Schematic diagram represents SAT technology. MBs are probe molecules that exhibit a characteristic stem-loop structure through which the 5′ and 3′ ends are maintained in close proximity. At this time, the fluorescence signals are quenched because the reporter groups and quencher groups are close to each other. The disruption of base-pairing in the stem, upon denaturation, allows the probe to hybridize to a complementary target. In this case, the reporter groups and the quencher groups are separated sufficiently, inducing the reporter groups to restore the fluorescence signal. Thus, the target can be determined based on the real-time increase in the fluorescence signal in the reaction system.

2.2 Reverse transcription (RT) amplification strategy: From RNA to DNA

The target RNA amplification strategy based on RT is introduce an additional RT process on the basis of the traditional isothermal amplification technology, that is, under the action of reverse transcriptase, complementary DNA (cDNA) is formed by RT according to RNA biological information, and then isothermal amplification techniques are used to produce a large amount of DNA products. Most isothermal amplification strategies, such as loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), cross primer amplification (CPA), rolling circle amplification, etc., can achieve a highly sensitive detection from RNA to DNA by introducing RT at the initial stage of the reaction. Next RT-LAMP, RT-RPA, and RT-CPA technologies are taken as examples for detailed introduction.

2.2.1 RT-LAMP

LAMP can achieve excellent specificity in a one-step reaction using a set of four target-specific primers, including a forward inner primer (FIP), backward inner primer (BIP), two outer primers described as forward outer primer (F3) and backward outer primer (B3), to recognize six distinct sites flanking the amplified DNA sequence [15,16].

As shown in Figure 3, the traditional LAMP technology can be divided into two phases, namely, the initial phase and the cycle phase [17]. In addition to its high specificity and sensitivity, the most significant advantage of LAMP is its ability to visually interpret results by observing changes in the turbidity of white precipitate of magnesium pyrophosphate, a byproduct of amplification [15].

Figure 3 
                     The schematic diagram of RT-LAMP for RNA detection. At the initial phase, cDNA strands formed by reverse transcription are used as templates for initiating reactions. F2 of FIP hybridizes to F2c of template DNA strand, guiding the synthesis of cDNA strand. The F3 primer anneals to the F3c region of template. With the help of DNA polymerase, starting the chain substitution reaction and releasing the FIP-linked complementary strand, double-stranded DNA is synthesized from the F3 primer and the template DNA strand. Because the complementary F1c and F1 regions exist at the chain of 5′ end of the released single strand, stem-loop structure is formed by complementary base. And then this single strand is regarded as a template in turn, and under the action of the BIP and B3, another new stem-loop structure at the other end of the DNA is assembled. A dumbbell structure is formed in the initial phase, this structure can serve as the template for cycle amplification during the subsequent reaction. Then, at the cycle phase, this dumbbell-like DNA structure was assembled repeatedly and amplified with the participation of FIP and BIP, the final products are stem-loop DNAs with several repeats of target and cauliflower-like structures with multiple loops.
Figure 3

The schematic diagram of RT-LAMP for RNA detection. At the initial phase, cDNA strands formed by reverse transcription are used as templates for initiating reactions. F2 of FIP hybridizes to F2c of template DNA strand, guiding the synthesis of cDNA strand. The F3 primer anneals to the F3c region of template. With the help of DNA polymerase, starting the chain substitution reaction and releasing the FIP-linked complementary strand, double-stranded DNA is synthesized from the F3 primer and the template DNA strand. Because the complementary F1c and F1 regions exist at the chain of 5′ end of the released single strand, stem-loop structure is formed by complementary base. And then this single strand is regarded as a template in turn, and under the action of the BIP and B3, another new stem-loop structure at the other end of the DNA is assembled. A dumbbell structure is formed in the initial phase, this structure can serve as the template for cycle amplification during the subsequent reaction. Then, at the cycle phase, this dumbbell-like DNA structure was assembled repeatedly and amplified with the participation of FIP and BIP, the final products are stem-loop DNAs with several repeats of target and cauliflower-like structures with multiple loops.

2.2.2 RT-RPA

RPA technology is a nucleic acid isothermal amplification strategy involving recombinase, single-stranded DNA (ssDNA) binding protein, strand displacement DNA polymerase, and two primer probes, which can amplify the template to detectable level within 3–10 min [18,19]. It has been widely used in biodefence [20], agriculture [21], water testing [22], and food testing [23], as well as medical diagnostics [24]. The schematic diagram is shown in Figure 4, double-stranded cDNA formed by RT can directly trigger subsequent RPA amplification [25,26]. The cyclic repetition of this process results in the exponential amplification of the target sequence [18].

Figure 4 
                     The schematic diagram of RT-RPA for RNA detection. Recombinase catalyzes the hybridization of primer with the homologous template sequence. The recombinase-primer filaments scan the target dsDNA and promote strand exchange at cognate sites. The resulting structures are stabilized by ssDNA-binding proteins to prevent primer displacement by branch migration. DNA polymerase recognizes the primer 3′ ends left by recombinase disassembly and initiates the primer extension reaction. The binding/extension events of two opposing primers generate one complete copy of the amplicon together with the original template.
Figure 4

The schematic diagram of RT-RPA for RNA detection. Recombinase catalyzes the hybridization of primer with the homologous template sequence. The recombinase-primer filaments scan the target dsDNA and promote strand exchange at cognate sites. The resulting structures are stabilized by ssDNA-binding proteins to prevent primer displacement by branch migration. DNA polymerase recognizes the primer 3′ ends left by recombinase disassembly and initiates the primer extension reaction. The binding/extension events of two opposing primers generate one complete copy of the amplicon together with the original template.

2.2.3 RT-CPA

CPA technology, developed by USTAR Biotechnologies (Hangzhou) LTD, is the first in vitro nucleic acid amplification technology with independent intellectual property rights in China, and has shown great potential in the scientific research of nucleic acid and the detection of transgenic crops [27,28]. The CPA amplification system mainly include cross primers, stripping primers, detection primers, and DNA polymerases with strand displacement activity. Depending on the number of cross primers in the system, this technique can be divided into two types: single crossing CPA and double-crossing CPA. The whole reaction is easy to operate and can be amplified at 63°C for 1 h to obtain a large number of single and double stranded products. The specific schematic diagram is shown in the Figure 5.

Figure 5 
                     The schematic diagram of RT-CPA for RNA detection. (a) Reverse transcription to form cDNA. (b) The process of single crossing CPA. A cross primer (2a1s), two stripping primers (4s and 5a) and two detection primers (2a and 3a) are required for the whole reaction process. The stripping primer (4s) is combined with the template region to guide the synthesis of DNA, thereby replacing the DNA strand guided by the cross primer with a single strand (5′-2a1s3s2s5s-3′), which can be combined with the 2a, 3a, and 5a to guide the synthesis of the DNA strand. At the same time, because the synthetic strand guided by the outer primer can replace the synthetic strand guided by the inner primer, two single-strand structures can be formed (5′-2a3a1a2s-3′ and 5′-3a1a2s-3′). The 2s region of the single-stranded DNA (5′-3a1a2s-3′) synthesized by 3a can combine with 2a and extend to form a double-stranded DNA product (Product 2). The 5′-1a2s-3′ region of the single-stranded DNA (5′-2a3a1a2s-3′) synthesized by 2a can be combined with the 2a1s to extend to form a double-stranded DNA product (Product 1). The two DNA strands of product 1, of which 5′-2a3a1a2s-3′ can combine with 2a and extend to form a new Product 1, while the other 5′-2a1s3s2s-3′ can combine with 2a and 3a again to form Product 1 and Product 2, respectively. Product 1 can participate in the next round of amplification cycle. (c) The process of double-crossing CPA. Two cross primers (2a1s and 1s2a) and two stripping primers (3s and 4a) are required for the whole reaction process. The 3s is combined with the template region to guide the synthesis of DNA, thereby replacing the DNA strand guided by the 2a1s with a single strand (5′-2a1s2s4s-3′), which can be combined with the 4a and 1s2a to guide the synthesis of the DNA strand (5′-1s2a1a2s-3′). DNA strand (5′-1s2a1a2s-3′) can be folded to form two stem-loop structures by the base complementary pairing principle. At the same time, the synthesized DNA strand (5′-1s2a1a2s-3′) can be combined with two cross primers, and one more binding site of cross primers can be generated in each step of the process, so as to generate a large number of mixed products of multi-branch single-stranded DNA and double-stranded DNA with various secondary structures.
Figure 5

The schematic diagram of RT-CPA for RNA detection. (a) Reverse transcription to form cDNA. (b) The process of single crossing CPA. A cross primer (2a1s), two stripping primers (4s and 5a) and two detection primers (2a and 3a) are required for the whole reaction process. The stripping primer (4s) is combined with the template region to guide the synthesis of DNA, thereby replacing the DNA strand guided by the cross primer with a single strand (5′-2a1s3s2s5s-3′), which can be combined with the 2a, 3a, and 5a to guide the synthesis of the DNA strand. At the same time, because the synthetic strand guided by the outer primer can replace the synthetic strand guided by the inner primer, two single-strand structures can be formed (5′-2a3a1a2s-3′ and 5′-3a1a2s-3′). The 2s region of the single-stranded DNA (5′-3a1a2s-3′) synthesized by 3a can combine with 2a and extend to form a double-stranded DNA product (Product 2). The 5′-1a2s-3′ region of the single-stranded DNA (5′-2a3a1a2s-3′) synthesized by 2a can be combined with the 2a1s to extend to form a double-stranded DNA product (Product 1). The two DNA strands of product 1, of which 5′-2a3a1a2s-3′ can combine with 2a and extend to form a new Product 1, while the other 5′-2a1s3s2s-3′ can combine with 2a and 3a again to form Product 1 and Product 2, respectively. Product 1 can participate in the next round of amplification cycle. (c) The process of double-crossing CPA. Two cross primers (2a1s and 1s2a) and two stripping primers (3s and 4a) are required for the whole reaction process. The 3s is combined with the template region to guide the synthesis of DNA, thereby replacing the DNA strand guided by the 2a1s with a single strand (5′-2a1s2s4s-3′), which can be combined with the 4a and 1s2a to guide the synthesis of the DNA strand (5′-1s2a1a2s-3′). DNA strand (5′-1s2a1a2s-3′) can be folded to form two stem-loop structures by the base complementary pairing principle. At the same time, the synthesized DNA strand (5′-1s2a1a2s-3′) can be combined with two cross primers, and one more binding site of cross primers can be generated in each step of the process, so as to generate a large number of mixed products of multi-branch single-stranded DNA and double-stranded DNA with various secondary structures.

3 Clinical application of RNA-based isothermal amplification technology in pathogen detection

3.1 Clinical application of transcriptional amplification strategy

Pathogen RNA detection based on transcription mediated isothermal amplification technology has been widely used in clinical, including respiratory pathogens, genitourinary pathogens, gastrointestinal pathogens, central nervous system infection pathogens, etc. Compared with other isothermal amplification detection strategies from RNA to DNA, direct transcription amplification technologies showing more direct and efficient advantages in RNA virus detection.

3.1.1 NASBA

NASBA technology was first proposed by Guatelli in 1990 [29], it shows outstanding advantages in time consuming, amplification efficiency, sensitivity, instrument cost, and other aspects, and has the characteristics of strong portability, high fidelity, good sensitivity, and strong openness. It is well suited for the detection of RNA in a total nucleic acid (with DNA and RNA) background. Deiman et al. comprehensively reviewed the amplification principle, reaction conditions, probe design essentials, products quantitative detection, and clinical application of NASBA technology [30]. This undoubtedly accelerates the construction of methodology based on NASBA. Morré et al. [31] compared the different primer sets for their sensitivities in NASBA, they developed 16S rRNA as an internal standard and shown that NASBA is a powerful amplification technique with a high sensitivity for the detection of Chlamydia trachomatis (CT). Mahony et al. [32] evaluated the clinical performance of the Basic Kit by testing a total of 250 specimens for Neisseria gonorrhoeae (NG) by culture and NASBA and a total of 96 specimens for CT by PCR and NASBA, the results proved that the NucliSens Basic Kit offer a versatile platform for the development of sensitive RNA detection assays for sexually transmitted diseases. The NASBA HIV-1 viral load determination test based on the electrochemiluminescence (ECL) test has been commercially available since 1995 and can be used to determine the viral load in the plasma of HIV-1 infected patients at the beginning and during antiviral therapy [33]. Beuningen et al. [34] developed a high throughput detection system for HIV-1 using real-time NASBA based on MBs, showing a sensitivity of 50 copies·mL−1. The improvement of this technology has dynamically increased the throughput of the test and shortened the hands-on time. With this assay, the amplification and real-time detection of 48 samples require only 90 min. Brink et al. [35] developed a new NASBA method for analysis of Epstein–Barr Virus (EBV) and compared with RT-PCR. Based on a lot of research works, we can easily know that NASBA has the advantages of rapid detection of RNA and accurate quantification in the background of DNA.

3.1.2 TMA

TMA and SAT technology are improved and developed on the basis of NASBA technology. Among them, TMA technology can be combined with a variety of signal detection methods to detect the amplification products, such as chemiluminescence, ECL, electrophoresis, colorimetry, etc., while the SAT technology mostly relies on fluorescent dyes or fluorescent group labeled detection probes to achieve real-time amplification and detection of targets.

Respiratory tract infection is an infectious disease caused by a variety of microorganisms including bacteria, viruses, mycoplasma, fungi, parasites, etc. [36]. It can occur throughout the year and at any age. The clinical symptoms vary with the degree of infection [37]. Early identification of the pathogen causing the infection and selection of a reasonable treatment plan can effectively prevent the abuse of antibiotics and enable patients to receive timely and effective treatment. TMA technology combined with downstream signal amplification methods such as fluorescence and chemiluminescence can be directly used to detect a variety of respiratory pathogens in throat swab or nasopharyngeal swabs, such as influenza A virus, influenza B virus, respiratory syncytial virus, human parainfluenza virus, adenovirus, Mycoplasma pneumoniae (MP), and Chlamydia pneumoniae. For example, the amplified RNA products by TMA are added to the microwell coated with the capture probe for hybridization, and the specific probe corresponding to the capture probe is added. At this time, a “three-cross-linked” composite structure of capture probe-RNA amplification product-specific probe will be formed. The other end of the specific probe is combined with a multi-biotin-labeled amplifying probe, and by adding a chemiluminescent substrate, the signal is amplified and detected on the chemiluminescence instrument. This technology combines rapid cell lysis and nucleic acid release to achieve accurate and qualitative detection of the abovementioned seven common respiratory tract infection pathogens within 2.5 h under constant temperature conditions.

TMA technology is also used to detect human papillomavirus (HPV) infection which is highly associated with cervical cancer. In 1989, Shah discovered that 99.7% of cervical cancers are related to HPV [38]. In 1995, World Health Organization pointed out that persistent infection of high-risk HPV is the main cause of cervical cancer. The etiology confirmed that the 14 high-risk types of HPV related to cervical cancer are: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 [39,40]. Persistent infection of high-risk HPV can increase the risk of cervical cancer by 250 times. Among them, high-risk HPV genotypes 16 and 18 are associated with about 80% of invasive cervical cancers [41]. Therefore, selecting a reasonable and effective screening method for rapid detection of high-risk HPV is an important method in screening high-grade cervical cancer-related lesions and the key to early detection of cervical precancerous lesions (CIN). HPV DNA tests can only detect HPV infection but cannot conclude whether it is a transient or a long-term/persistent HPV infection [42]. HPV E6/E7 messenger ribonucleic acid (HPV E6/E7 mRNA) tests can be conducive to the judgment of whether HPV virus infection is in the active phase, and can provide a new method with more clinical reference value to those patients who were tested positive by HPV DNA but not necessary to make colposcopic referral [43,44]. Many studies associate E6/E7 mRNA positivity with an integration of the progression of carcinogenesis, revealing an association between the degree of integration, histological, and cytological lesion severity [45,46]. By prospective comparison, Dabeski et al. [47] concluded that HPV E6/E7 mRNA testing is more specific and has a higher positive predictive value than HPV DNA testing, and that viral oncoproteins E6 and E7 are superior biomarkers for the detection of high-risk HPV-associated squamous intraepithelial lesions of the uterine cervix. At present, TMA combined with hybrid protection assay is commonly used in clinical to detect high-risk HPV E6/E7 mRNA (Aptima HPV Assay). Ratnam et al. [48] compared the Aptima and Hybrid Capture 2 DNA test (HC2) for detecting CIN 2+ in referral population, and concluded that the Aptima is as sensitive as HC2 but more specific for detecting CIN 2+, and it can serve as a reliable test for both primary cervical cancer screening and the triage of borderline cytological abnormalities. Ge et al. [49] mentioned that the expression of E6/E7 mRNA was significantly increased after HPV DNA integration, resulting in higher specificity and positive predictive value of the Aptima method for HPV E6/E7 mRNA detection than that of the Cobas for HPV DNA detection. Dockter et al. [50] concluded that the APTIMA HPV assay showed excellent performance and robustness in analytical sensitivity, analytical specificity, and low variability, which is a better indicator of disease progression than detection of the presence of HPV DNA.

Furthermore, Chernesky and Jang [51] reviewed the TMA assays for CT and NG, they mentioned that TMA has the greatest sensitivity of all the commercial nucleic acid amplification tests for the diagnosis of infections from noninvasive samples, such as in urine.

3.1.3 SAT

Of course, SAT technology is also directly used in the detection of respiratory infectious diseases. Li et al. [52] evaluated the potential application value of SAT technology in the diagnosis of MP infection, and through comparison they found that SAT-MP assay was more sensitive to M. pneumoniae than real-time PCR with a detection limit of 101 CFU·mL−1. They mentioned in the research that the superior sensitivity may be attributed to higher level of rRNA than DNA at the same concentrations of M. pneumoniae. Moreover, the SAT-MP has more efficient amplification kinetics and desirable specificity in identifying M. pneumoniae than PCR. Since the effective diagnosis of M. pneumoniae pneumonia (MPP) in children has been hampered by the difficulty in achieving an early diagnosis, Li et al. [53] have used antibody results as the evaluation standard to compare the advantages of SAT technology. They concluded that SAT is an effective diagnostic tool for rapid, sensitive, and specific detection MPP at the initial stage of an infection. SAT is correlated with the clinical symptoms and therapeutic effect of MPP and can be used as an indicator to evaluate the therapeutic effect and assist the diagnosis of recovery, so as to prevent excessive medical treatment. SAT technology can also be successfully used to detect Mycobacterium tuberculosis (TB) infection and the corresponding drug resistance gene [54,55,56]. Li et al. [57] applied SAT method to accurately and rapidly detect extrapulmonary tuberculosis (EPTB) and demonstrated that SAT is a simple and rapid method with high specificity and clinical diagnosis value which may enhance the detection of EPTB. Fan et al. [55] concluded that the SAT-TB assay can be widely applied in the clinic, and it is an asset in the early detection and management of PTB suspects, especially in those patients who are smear negative or sputum scarce. In addition, SAT technology has been applied for the detection of novel coronavirus SARS-CoV-2 by automated instruments, realizing the full automatic detection of “sample in and result out” in only 90 min, which greatly shortened the detection time, saved the labor force, and reduced the potential infection risk of manual open operation.

After genital tract pathogens invade the body, they can cause inflammation of the genitourinary system such as cervicitis, pelvic inflammatory disease, prostatitis, and epididymitis. If it is ascending infection of the upper genital tract, it will cause many serious consequences, such as infertility, miscarriage, premature delivery, premature rupture of membranes, etc. [58]. It will also cause respiratory and urinary diseases such as neonatal pneumonia and conjunctivitis through mother-to-child transmission. The method of microbial culture has a certain risk of false negatives, and it takes a long time, usually 3–5 days. At present, the most commonly used SAT in clinical practice is not limited by sample types. It can detect four common genitourinary tract infection pathogens in cervical secretions, vaginal secretions, urine, semen, throat swabs, and even breast milk within 1.5 h. The detected target pathogens are Ureaplasma urealyticum (UU) RNA, CT RNA, NG RNA, and Mycoplasma genitalium (MG) RNA [59,60]. The target RNA product amplified by the SAT technology and the real-time fluorescent signal is detected by the detection instrument. The change in Ct value of fluorescence curve can dynamically reflect the changes in pathogen load and the clinical treatment effect. The biggest advantage of SAT in detecting RNA is that it can be detected with urine samples. Taking CT in urine as an example, the PCR method requires to centrifuge the urine to obtain bacterial precipitation before detection. If the CT content in urine is small, its DNA target will not be detected due to the lower sensitivity of PCR detection. However, because the content of rRNA is thousands of times higher than that of pathogens, direct cleavage of urine can release a large amount of rRNA, so that the lysate contains enough rRNA for SAT detection. Therefore, SAT method has the advantages of high sensitivity and can use urine samples for detection. By comparing with the culture method, Zheng et al. [61] drew the conclusion that SAT is a rapid and accurate method for detecting UU infection in semen samples, with a higher sensitivity and accuracy, and it can also be used to evaluate the therapeutic effects. Liang et al. [62] compared the RNA-based and DNA-based nucleic acid amplification methods for the detection of CT, NG, and UU in urogenital swabs. They also demonstrated that the SAT assay has more excellent agreements and higher sensitivities than qPCR assay, so it is a better choice for screening, diagnosis, and surveillance of sexually transmitted diseases. Kirkconnell et al. [63] evaluated the analytical and clinical performances of SAT for MG RNA detection.

Enteroviruses belong to the enterovirus genus of the Picornaviridae family, including poliovirus, Coxsackievirus, and orphan virus (ECHO virus) that causes intestinal cytopathy in humans. The high-risk groups of infection are mainly infants and young children, especially those under 3 years old are more susceptible to enterovirus infection. This type of pathogen infection usually occurs in spring and summer, with maculopapular rash and herpes on the hands, feet, and oral cavity as the main manifestations, namely, hand, foot, and mouth disease (HFMD) [64]. In severe cases, it can cause encephalitis, encephalomyelitis, meningitis, pulmonary edema, circulatory failure, etc. At present, RNA isothermal detection for enteroviruses mostly relies on SAT technology. Chen et al. [65] developed and evaluated a SAT technology for detection of enterovirus 71 (EV71) and incorporated an RNA internal control (IC) system. They found that the SAT-EV71 with IC developed in this study is highly specific and sensitive in the detection of EV71 from clinical specimens. Xu et al. [66] developed a new assay called SAT-CA16 for the detection of Coxsackievirus A16 (CA16) RNA, and they established that the sensitivity of SAT-CA16 for detecting clinical specimens was higher than that of real-time PCR. Chen et al. [67] established a method called SAT-EV/SAT-CA16 with IC for the detection of EV/CA16 from clinical specimens which took only about 1 h, it provides a practical platform for the clinical laboratory diagnosis and rapid detection of EV or CA16 in outbreak situations of HFMD.

3.2 Clinical application of RT amplification strategy

By introducing additional RT process into the traditional DNA-based isothermal amplification technology, the efficient DNA amplification strategy targeting RNA can be realized. Yaqing et al. [68] developed and validated a one-step, single-tube RT-LAMP assay for the detection of human EV71. They designed primer probes against the same target site to compare the parallel sensitivities of RT-LAMP, RT-PCR, and real-time RT-PCR, the results demonstrated that the detection limit of RT-LAMP assay was 160 copies in samples after RNA extraction, which was 10-fold higher in sensitivity than the traditional RT-PCR. Yan et al. [69] developed and evaluated a method, namely, RT-LAMP combined with lateral flow device technology to rapidly detect CA16 and showed 100% specificity in clinical specimens. Arita et al. [70] demonstrated that RT-LAMP showed a higher sensitivity comparable to that of the cell culture for the detection of poliovirus, and human enterovirus species A and C directly from stool samples. By double-stranded DNA binding fluorescent dye, Shi et al. [71] developed a visual method based on RT-RPA to directly detect EV71 under the UV lamp. The method has accurately detected 123 clinical samples and demonstrated excellent sensitivity. Yin et al. [72] established RT-RPA assay to detect EV71 subgenotype C4 in clinical sample and showed satisfactory sensitivity and specificity. Combined with RT-RPA and lateral flow strip, Xie et al. [73] established a visualization method for the detection of Coxsackievirus A6 (CA6) in less than 35 min at 37℃ without expensive instruments.

4 Summary and outlook

RNA-based isothermal amplification technology cannot replace PCR technology in pathogen detection, but it makes up for the shortage of traditional PCR detection technology such as depends on instruments and takes a long time. It has great development potential and unique application prospect in the field of rapid detection, full-automatic integrated detection, and other aspects. It is the future development direction of nucleic acid detection technology for infectious pathogens. However, the available RNA detection technologies for the purpose of point-of-care diagnostics of infectious pathogens in resource-poor areas and on-site are extremely limited. To realize the field application of nucleic acid isothermal amplification technology, it is also necessary to consider the miniaturization of instruments or visual interpretation without instruments and acceptable detection cost. As a new reaction and detection carrier, microfluidic chip is expected to be combined with different isothermal amplification reactions to develop a series of compact and portable point-of-care detection platforms.

In addition to RNA detection technology based on isothermal amplification, with the continuous improvement of analytical techniques, complete transcriptome analysis at the RNA level will no longer be a blank. The basic theory of the central dogma states that RNA is the hub for the transmission of genetic information, and if there is some kind of abnormality in the RNA transcript, it indicates that the abnormality is caused by DNA mutation. Careys (America) is one of the first companies to conduct research and development of this type of technology. Through the evaluation of the entire transcriptome, it can deeply study RNA-level variation, discover, and detect gene fusion, gene splicing variation, and expression changes. This technology perfectly fits the needs of precision medicine and has far-reaching application prospects. However, conditions such as high technical difficulty, high detection cost, and harsh instrument requirements limit its current development speed.

  1. Funding information: This research was funded by Cadres Healthcare Research Projects in Sichuan Province (No. 2021-1703) and Clinical Discipline Development Fund of West China Second Hospital, Sichuan University (No. KL066).

  2. Author contributions: Jie Teng and Li Chang: performed the methodology, investigation, and wrote the original draft; Guanglu Che and Qiuxia Yang: provided software services, supervised, and collected the clinical data; Yuan Tan and Jiaxin Duan: performed the literature analysis and reviewed the manuscript. Jie Teng and Shuyu Lai: supervised the work, analyzed the data, and wrote, reviewed, and edited the manuscript. Yongmei Jiang: acquired funding and administrated the projects. All authors have read and agreed to the published version of the manuscript.

  3. Conflict of interest: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article.

  4. Data availability statement: All data generated or analyzed during this study are included in this published article.

Appendix

Table A1

Clinical application of transcriptional amplification strategy in RNA detection

Platform Method Advantage Performance Duration of detection Condition of reaction Application Reference
Electrophoresis NASBA Includes 16S rRNA internal standard; suitable and sensitive The detection limit is 10−3 IFU 125 min 41°C and 45°C CT [31]
ECL NASBA High sensitivity and specificity Sensitivity of 1 IFU for CT and 1 CFU for NG 140 min 41°C CT, NG [32]
Fluorescence NASBA Rapid, real-time, with high sensitivity and specificity Sensitivity of 50 copies·mL−1 45 min 41°C HIV-1 [34]
Electrophoresis NASBA Realized the detection of clinical samples Sensitivity of 100 copies·mL−1 120 min 41°C EBV [35]
Chemiluminescent TMA Excellent performance and robustness in analytical sensitivity, analytical specificity, and low variability Linear range from 100 to 240 DNA copies·mL−1 180 min 42°C HPV E6/E7 mRNA [50]
Fluorescence SAT Compared with PCR, it has higher sensitivity, more efficient amplification kinetics, and better specificity identification Linear range from 101 to 107 CFU·mL−1 40 min 42°C MP [52]
Fluorescence SAT Accurate, cheap, and rapid The sensitivity, specificity, and accuracy are 75.8%, 100%, and 80.2%, respectively 120 min 42°C TB and the corresponding drug resistance gene [55]
Fluorescence SAT Accurate and rapid The sensitivity and specificity are 87.7% and 99.4% in urine sample, respectively 90 min 42°C UU, CT, NG, MG [62]
Fluorescence SAT Highly specific, sensitive, and rapid Linear range from 105 to 101 copies·mL−1 80 min 60°C and 42°C EV71 [65]
Fluorescence SAT A rapid, sensitive, specific, reliable, and promising method The sensitivity and specificity were 100% and 99.2%, respectively; the detection limit was 0.1 FFU·mL−1 80 min 60°C and 42°C CA16 [6]

CT: Chlamydia trachomatis; NG: Neisseria gonorrhoeae; UU: Ureaplasma urealyticum; MG: Mycoplasma genitalium; HIV: human immunodeficiency virus; EBV: Epstein–Barr virus; HPV E6/E7 mRNA: HPV E6/E7 messenger ribonucleic acid; MP: Mycoplasma pneumoniae; TB: Mycobacterium tuberculosis; EV71: Enterovirus 71; CA16: Coxsackievirus A16.

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Received: 2022-09-04
Revised: 2022-11-19
Accepted: 2022-11-29
Published Online: 2022-12-23

© 2022 the author(s), published by De Gruyter

This work is licensed under the Creative Commons Attribution 4.0 International License.

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