Sequence-Specific Probe-Mediated Isothermal Amplification for the

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Sequence-Specific Probe Mediated Isothermal Amplification for the Single-Copy Sensitive Detection of Nucleic Acid Xin Ye, Xueen Fang, Yang Li, Lijuan Wang, Xinxin Li, and Jilie Kong Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00812 • Publication Date (Web): 03 May 2019 Downloaded from http://pubs.acs.org on May 3, 2019

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Analytical Chemistry

Sequence-Specific Probe Mediated Isothermal Amplification for the Single-Copy Sensitive Detection of Nucleic Acid

Xin Ye, a Xueen Fang, *a Yang Li, bc Lijuan Wang, bc Xinxin Li, bc and Jilie Kong *a a. Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, P.R. China. b. Shanghai Suchuang Diagnostic Products Co., Ltd Shanghai 201318, P. R. China. c. Shanghai Suxin Biotechnology Co. Ltd Shanghai 201318, P. R. China.

*Correspondence

should

be

addressed

to

X.E.F.

mail:[email protected]) or J.L.K. (E-mail:[email protected])

ACS Paragon Plus Environment

(E-

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract There is currently lacking method for precisely monitoring the progress of isothermal amplification reactions by means of sequencespecific fluorescent probes like TaqMan probe used in PCR system. Here, we created a circular fluorescent probe mediated isothermal amplification (CFPA) method. This novel method uses two circular fluorescent probes and Bst DNA polymerase to construct an overlapping structure that can be cut off by flap structure-specific endonuclease 1, separating the fluorescence and quenching groups on the probes. The results showed single-copy sensitivity, ultrahigh specificity, stability (C.V. 0.05). In summary, we present a new, reliable and precise isothermal amplification approach for applications in biomedical research and the clinical accurate diagnosis of pathogen infections.

Keywords: flap structure-specific endonuclease 1; isothermal amplification; molecular diagnosis; nucleic acids


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Introduction Nucleic acid detection is a widely used and revolutionary technique in the field of biomedical research that enables precision clinical diagnoses of infectious pathogens and genetic diseases. It is universally acknowledged that polymerase chain reaction (PCR), established in 1986 1,

is the most basic and reliable nucleic acid detection method. Over the

past few decades, major progress has been made with the sensitivity, specificity and quantitative ability of PCR being dramatically improved by using double-stranded DNA intercalating molecules 2 such as SYBR Green I and Eva Green (for relative quantification), fluorophore-labeled oligonucleotides such as TaqMan probes 3 (for quantification) and digital PCR methods 4 (for absolute quantification). The advantages are obvious; however, PCR still has some limitations. One major limitation is the need for specialist equipment and skilled operators, which severely restricts its application particularly in resource-limited regions and in some point-ofcare settings 5. Various isothermal nucleic acid amplification methods have been developed to optimize PCR systems 6. Among these, representative methods include recombinase polymerase amplification 7, loop-mediated isothermal amplification (LAMP) 8, strand displacement amplification 9, helicase-dependent amplification

10

and cross priming amplification

11.

Most of these methods utilize the Bst DNA polymerase, large fragment,



which has strong strand displacement activity allowing for cyclic amplification of target sequences without the need for thermal cycling. These isothermal amplification methods have low-level requirements in terms of specialist equipment and are therefore more suitable for use in remote areas and point-of-care settings 12-14. Several approaches have been employed to monitor the isothermal amplification process and detect amplified products, the most common and classic methods include high affinity nucleic acid binding dyes (SYBR Green or SYTO), pH-sensitive dyes (neutral red), turbidity or electrophoresis 15. Notably, these methods are all based on nonspecific target sequence recognition and therefore nonspecific signals as well as lower sensitivity limit their widespread application16,17. As mentioned above, the integration of a fluorescent probe like TaqMan probe into a PCR system is promising in terms of specific target sequence recognition, which may lead to increased specificity and sensitivity. However, due to the lack of cutting activity by the Bst DNA polymerase, large fragment

18,

commonly used to trigger the isothermal

amplification, it is hard to realize a highly specific and sensitive fluorescent probe-mediated reporter for monitoring the isothermal amplification process. To establish a fluorescent probe mediated sequence-specific recognition based high sensitivity and specificity isothermal amplification approach, an enzyme with cutting activity was needed to be coupled with


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the Bst DNA polymerase to synthesize DNA and separate the fluorescent and quenching groups on the probe when a target sequence was encountered. Furthermore, the reaction temperature and buffer conditions of this enzyme must be close to that of the Bst DNA polymerase. Based on these requirements, the thermostable flap endonuclease (FEN1) was found to be suitable. FEN1 is a structure-specific endonuclease that cleaves single-stranded DNA or RNA at the bifurcated end of a base-paired duplex19. It can specifically recognize at least a one-base overlapping structure formed by two probes (forward and reverse) hybridizing to adjacent sequences in a target DNA and trigger the cleavage of the 5ʹ flap of the probe 20,21. In this study, based on the FEN1 enzyme and Bst DNA polymerase, we constructed a circular fluorescent probe and developed a novel isothermal nucleic acid amplification method (CFPA) for the specific, sensitive and rapid detection of pathogens.

Experimental section Regents and equipment A series of Bst DNA polymerases including full length (M0328), large fragment (M0275), 2.0 (M0537), 2.0 warmstart (M0538) and 3.0 (M0374), along with the supplementary reaction buffers and the thermostable flap endonuclease, FEN1, (M0645) were purchased from New England Biolabs. Regular agarose (111860) was purchased from BIOWEST. The ethidium ACS Paragon Plus Environment


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bromide (A500328), DNA marker (B600333), 6×ficoll gel loading buffer (B540084) and 50×TAE buffer (B548101) were purchased from Shanghai Sangon

Biotech.

UltraPure™

DNase/RNase-free

distilled

water

(10977015) was purchased from ThermoFisher Scientific. The nucleic acid extraction

kit

(SC-702)

was

purchased

from

Shanghai

Suxin

Biotechnology. The cDNA was synthesized using PrimeScript™ RT master mix (RR036A) from Takara Biomedical Technology. The detection of fluorescent signals during the amplification was based on the LineGene 9600 PCR system (Hangzhou Bioer Technology). The concentration of the extracted nucleic acid was measured using a micro-volume spectrophotometer (SMA4000). The gel electrophoresis cell (HE-90) and gel imaging system (Tanon 3500) were from Shanghai Tanon Science & Technology. The plasmid carrying the highly reversed sequences of the targets (rotavirus and astrovirus) in the present study was synthesized by Takara Biomedical Technology. To obtain the standard RNA samples, the rotavirus plasmids were subjected to in vitro transcription using RNA polymerase, which was also synthesized by Takara Biomedical Technology.

Design of the circular fluorescent probe For each target, a forward and reverse circular fluorescent probe (CFP)


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each targeting four regions upstream and downstream of the target were designed, to ensure specific binding. Each probe was labeled with a fluorescent group at the 5ʹ end and a quenching group at the T base in around the tenth position. The fluorescence could be detected only when the fluorescent and quenching groups were separated. The optimized sequences of the forward and reverse CFPs for rotavirus and astrovirus are listed in Table S1. All probes used in the present study were synthesized by Shanghai Sangon Biotech and purified by high-performance liquid chromatography.

The CFPA system Firstly, the concentrations of probes (Figure S1), types of DNA polymerase among the Bst family (Figure S2), the amount of FEN1 (Figure S3) and the reaction temperature (Figure S4), were systematically optimized. The optimum assay conditions were used for this study and were as follows: the total volume of the amplification system was 12.5 μL comprising: 1.2 μΜ of each forward and reverse CFP, 1×isothermal amplification buffer (B0537), 6 mM MgSO4, 1.4 mM dNTP mix, 4 U Bst 2.0 warmstart DNA polymerase, 0.6 U FEN1, 2.5 μL nucleic acid template and UltraPure water. The system was incubated at 63 oC for 60 min and the fluorescent signal was detected every minute. The time to threshold (Tt)



was defined as the time to reach a fluorescent threshold that was automatically calculated by the software.

Clinical sample collection and nucleic acid extraction All clinical samples (stool samples) were collected from children with diarrhea diagnosed in the central laboratory at the Children’s Hospital at Fudan University. All patients were infected by a single pathogen: rotavirus (n=96), astrovirus (n=21), norovirus (n=30), adenovirus (n=19), Clostridium difficile (n=3), Salmonella enteritidis (n=3) or Campylobacter jejuni (n=2). The above-infected pathogens (n=78) except rotavirus were used as negative samples in the clinical trials. Informed consent was obtained from all subjects. All raw stool samples were stored at -80 oC. The total nucleic acids including DNA and RNA were extracted from the raw stool samples according to the manufacturer's instructions. Total nucleic acid was then divided into two tubes. One was stored directly at -80 oC until use, the other was reverse transcribed to cDNA according to the manufacturer's instructions and stored at -20 oC until use.

Electrophoresis and sequencing The amplified products from the CFPA method were subjected to electrophoresis using 2% regular agarose in 1×TAE buffer and gels were stained with ethidium bromide. Electrophoresis was performed at a


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constant voltage of 100 V for 40 min. The loading volume in each well on the gel was 6 μL, into which 2.5 μL of amplified product, 2.5 μL of UltraPure water and 1 μL of 6×loading buffer was added. After electrophoresis, the bands on the gel were extracted for further TA cloning sequencing analysis to verify the presence of the desired products.

Statistical analysis For comparison of the CFPA assay to the golden standard method (using human rotavirus real time PCR kit, JC50101 purchased from Jiangsu Bioperfectus Technologies Company, Ltd), clinical trials were conducted. A minimum sample size of 74 infected and uninfected cases were calculated based on expected sensitivity and specificity levels of 95%, respectively, for assessment of the accuracy of the new CFPA method using MedSci software based on previously published work,22 and the number of samples collected met this requirement. Receiver operating curves (ROCs) were drawn using MedCalc statistical software to analyze the diagnostic accuracy of the new CFPA method.23 The area under the ROC curve (AUCROC) was compared as described by DeLong and coworkers.24 A test was considered significant if the p-value was less than 0.05.

Results and discussion



The mechanistic basis of the CFPA method is illustrated in Scheme 1. Briefly, two CFPs comprised the primer system, which acted both as an extending primer and a sequence-specific reorganization probe. More specifically, the forward and reverse probes recognized sequences upstream or downstream of the target, respectively. The 5ʹ end of each probe was labeled with a fluorescent group and nearby was a quenching group. Firstly, utilizing the DNA polymerization and strand displacement ability of Bst DNA polymerase, the forward CFP matched with the target and extended to form a circular structure on the side of the chain with a quenched fluorescent group at its 5ʹ end. Then, the reverse CFP matched with the target and extended. When it encountered the 5ʹ end of the circular structure formed in the last step, it formed an overlapping structure that was recognized by FEN1. Consequently, FEN1 exerts its cleavage activity to separate the fluorescent and quenching groups in the 5ʹ region of the overlapping structure, forming detectable fluorescent signals. Similarly, the fluorescent group in the reverse CFP could also be cleaved by the forward CFP in the same way as described above, which could double the detectable fluorescent signals. Several benefits arise from using this method. First, the reaction proceeds under isothermal conditions, which is convenient. Second, the forward and reverse CFP needs to recognize four parts upstream and downstream of the target area. The overlapping structure can only be


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generated when all four parts of the sequence match. This ensures that the detectable fluorescent signal is target sequence-specific and consequently the method has extremely high specificity compared with traditional double-stranded DNA intercalating dyes (SYBR Green, ethidium bromide or SYTO). Third, the CFP used here showed high sensitivity in the present method. These characteristics indicated the great potential of the present CFPA method to be used for nucleic acid detection, especially for clinical diagnoses in laboratories and point-of-care settings.

Scheme 1. Mechanistic basis of the novel circular fluorescent probe mediated isothermal nucleic acid amplification (CFPA) method

The present CFPA method was preliminarily verified for the detection of pathogens in stool samples from children with diarrhea. The pathogens in these samples comprised mainly of viruses, particularly rotavirus and astrovirus

25,26.

Plasmids containing the conserved gene of rotavirus and ACS Paragon Plus Environment


astrovirus were constructed for verification of the feasibility of this method. The results, shown in Figure 1, revealed that both rotavirus and astrovirus plasmids showed a typical fluorescence amplification curve that comprised a baseline period, exponential amplification period and a platform period. Water was used as a negative control for each assay and no fluorescent signal was detected for either target (Figure 1A, B, the two amplification curves of each sample were the results of the duplicate experiment). In this study, we compared the fluorescence amplification curve using two fluorescent-labeled probes, a single fluorescent-labeled probe and an unlabeled probe, respectively. The results in Figure 1C clearly show that the fluorescent signal during the platform period for the two fluorescentlabeled probes was about twice that obtained using a single fluorescentlabeled probe. This indicated that the fluorescent group on the reverse CFP doubled the fluorescent signal, strongly supporting the mechanism of CFPA proposed above. Furthermore, the specific amplified products were further analyzed by agarose gel electrophoresis and the results are shown in Figure 1D. Only positive samples could generate obvious bands on the gel, whereas the negative samples did not generate any bands, which was consistent with the results of the fluorescence amplification curves. In addition, the specific CFPA products of rotavirus were further analyzed by TA cloning and sequencing, and the results were consistent with expectations (Figure


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1E). Taking the above data into account, we can preliminarily determine the feasibility and reliability of the CFPA method. The distribution pattern of the products on the gel was different from that obtained with the classical PCR method but was similar to those of other Bst DNA polymerase mediated isothermal amplification methods such as LAMP 8, strand exchange amplification 27 and cross priming amplification 11, which present a ladder repeat structure, a pattern characteristic of Bst DNA polymerase, as described by previous studies 28,29.

Figure 1. Verification of the mechanism of the present CFPA method. (A) and (B) the fluorescence amplification curves when targeting rotavirus (RV) and astrovirus (AS), respectively; (C) the fluorescence amplification curves when using double or single fluorescent-labeled probes, respectively; (D) electrophoresis pattern of the amplified products (line M refers to marker, line 1 and 2 were RV plasmid, line 3 and 4 were AS plasmid, line 5 was H2O using RV CFPs and line 6 was H2O using AS CFPs); (E) TA cloning and sequencing results of the band from line 1 in the gel.



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Then, a total of 32 DNA samples (among them, 8 samples were reverse-transcripts from rotavirus cDNA, 8 norovirus-infected, 8 astrovirus-infected, and 8 adenovirus-infected samples) were used to assess the detection performance of the CFPA for DNAs. The results in Figure 2A and B clearly show that our CFPA method had 100% specificity and sensitivity, giving a completely accurate diagnosis when detecting the above samples. Such precise results would allow physicians to choose the most appropriate and effective drugs for particular patients. We used serially diluted rotavirus plasmid samples (from 1×107–1×101 copies) to determine the detection limit and linearity of the CFPA method. The results in Figure 2C and D demonstrated a good linear relationship between the Tt value and the logarithmic value of the plasmid concentration (range from 1×107–1×101 copies, R2=0.97608). Next, the plasmid sample was continuously diluted until a single-copy, and the fluorescence amplification curves (Figure 2E) and electrophoresis results (Figure 2F) all demonstrated that our CFPA method could achieve single-copy sensitivity. Although digital isothermal amplification method

30,31

could also achieve

this ultrahigh sensitivity, but the costs and the equipment requirements were much higher than the present CFPA method. These findings indicated that our method could be used to detect low concentrations of infecting pathogens, minimizing the misdiagnosis rate when applied in clinical settings. The good linear relationship indicated the


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potential quantification ability of the CFPA method, broadening its potential applications. Besides the cDNA of rotavirus, the cDNA of astrovirus was also used to assess the specificity, sensitivity, detection limit and linearity of the CFPA method (Figure S5). All of the results indicated the excellent performance of our CFPA method for the analysis of DNAs.

Figure 2. The specificity, sensitivity, detection limit, linearity and the single-copy sensitivity of the CFPA method for the cDNA of rotavirus (A–F). The fluorescence amplification curves when detecting rotavirus cDNA from infected (A) and uninfected (B) samples; (C) the fluorescence amplification curves of serially diluted cDNA (from 1×107–1×101 copies); (D) the linearity between the Tt value and logarithmic value of rotavirus plasmid concentrations; the fluorescence amplification curves (E) and electrophoresis pattern (F) of the samples diluted until a single-copy. Many viral pathogens possess RNA genomes, therefore ideally nucleic

acid detection methods would be able to simultaneously detect DNA and RNA for effective clinical application 32. Methods previously employed for detecting RNA targets include RT-PCR, RT-RPA and RT-LAMP33, which ACS Paragon Plus Environment


normally involve an extra step of reverse transcription, in which RNA is reverse transcribed into the cDNA strand using an additional reverse transcriptase 34. We also incorporated this step into our method in the above CFPA test. In the present study, Bst 2.0 warmstart DNA polymerase was found to be optimal for our method. This type of enzyme possesses DNA polymerase activity, strand displacement activity and innate reverse transcriptase activities, based on which, one step RNA detection could be realized using our CFPA method

35.

We employed rotavirus, a double-

stranded RNA virus that is the most frequent viral pathogen causing diarrhea among children and leading to serious symptoms and a poor prognosis 36, to evaluate the performance of the CFPA method. The results in Figure 3A and B reveal typical fluorescent amplification curves when rotavirus RNA was present in the clinical samples. This supported our hypothesis that the CFPA method could directly detect RNA samples without the need for an extra reverse transcription step. The CFPA method provided 100% sensitivity and specificity in the detection of 8 rotavirusinfected samples and 24 rotavirus-uninfected samples without a reverse transcription step, which was similar to the performance in detecting cDNA samples. In addition, our CFPA method could also be applied for the detection of single-stranded RNA (ssRNA), which was verified using astrovirus infected and uninfected samples (Figure S6).


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Then, the in vitro transcript rotavirus RNA samples were used to assess the detection limit and linearity. It was shown that the detection limit for RNA samples remained as low as 10 copies (Figure 3C), and the linearity was good between the Tt value and the RNA concentration (Figure 3D). To achieve the quantitative function of a nucleic acid detection method, the stability and reproducibility of the assay are important. We, therefore, chose three concentrations of the RNA samples to evaluate the quantitative performance of the CFPA method. The results presented in Figure 3E and F clearly show the good reproducibility of the Tt values among the eight replicate experiments with a small coefficient of variation (C.V.) of less than 0.1 for each of the three sample concentrations (representing high, medium and low), which strongly indicated the potential quantitative ability of CFPA for the RNA samples.



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Figure 3. The specificity and sensitivity of the CFPA method for the direct detection of RNA. The templates were rotavirus-infected RNA (A) and uninfected RNA (B) samples; the detection limit (C) and linearity (D) of the CFPA method when detecting rotavirus RNA samples; (E) the reproducibility of the Tt values among eight replicate experiments using three different RNA sample concentrations (representing high, medium and low concentrations); (F) the SD and C.V. values of these replicate experiments for the three sample concentrations.

To verify the practical clinical application of the CFPA, we performed systematic clinical trials for the diagnosis of rotavirus infection, compared with the gold standard method (commercial PCR kit), for 174 clinical samples including 96 rotavirus-infected samples and 78 rotavirusuninfected samples (negative samples). As shown in Table 1, our CFPA method showed 96.88% sensitivity and 100% specificity for the detection of rotavirus, demonstrating the promising potential of our method in clinical applications. Table1. Results of clinical trials of the comparison between CFPA and PCR PCR

CFPA Total

Positive

Negative

Total

Positive

93

0

93

Negative

3

78

81

96

78

174

Next, receiver operating curves (ROCs) were constructed to determine the diagnostic accuracy of the CFPA assay based on the above results. This revealed that the CFPA method was able to discriminate


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between rotavirus infected and uninfected samples with an AUCROC of 0.984; p0.05) (Figure 4B).

Figure 4. Receiver operating curve (ROC) analysis of the CFPA method for the diagnosis of rotavirus infection (A) and a comparison of the CFPA and PCR methods for the diagnosis of rotavirus infection (B).

In practical applications of nucleic acid based diagnosis, there exist various endogenous and exogenous interfering substances that may inhibit the nucleic acid amplification process and generate false negative results. In this study, we chose various interfering substances, including druginduced exogenous substances and endogenous substances such as hemoglobin, to evaluate the anti-interference ability of the CFPA method. The concentration of these interfering substances was at the maximum



clinical dose. The results in Figure 5 clearly show that most of these interfering substances only had a small effect on the CFPA method and could not totally inhibit the CFPA method in detecting three independent samples. This finding broadens the potential clinical applications of our method for monitoring treatment effects and providing accurate diagnoses.

Figure 5. The anti-interference ability of the CFPA method. Each column means the Tt value in the presence of different interfering substance.

In addition, we also compared the diagnosis performances of the present CFPA method with a classic isothermal amplification methodLAMP and the results were shown in Table S2. When using LAMP method to diagnosis the above 174 clinical samples, the sensitivity and specificity were 93.75% and 57.69% respectively. These two analysis parameters were not as good as CFPA, especially the poor specificity. These results confirmed the significant advantages of CFPA over the representative isothermal amplification methods-LAMP.


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Conclusions In summary, we first developed a sequence-specific circular fluorescent probe mediated isothermal nucleic acid amplification (CFPA) method using Bst 2.0 warmstart DNA polymerase and FEN1. The mechanistic basis of this method was confirmed. This new method could realize nucleic acid (both DNA and RNA) detection with single-copy sensitivity, ultrahigh specificity, reproducibility and anti-interference ability, owing to the advantages of both the common isothermal amplification method and the fluorescent probe method. Furthermore, in the future, the CFPA could integrate with other technology like microfluidic to develop portable and automated nucleic acid analysis equipment, which will greatly promote its application in the field of pointof-care detection. Overall, this CFPA method possesses excellent performance in practical applications for the accurate clinic diagnosis of pathogen infections.

Conflicts of interest There are no conflicts to declare.

Acknowledgements This work was supported by the National Key R&D Program of China



(2017YFA0205100), the National Natural Science Foundation of China (21427806, 21175029, and 21335002), the Introduce Talents of Fudan University Research Funding (JIH1615032), and the Shanghai Leading Academic Discipline Project (B109).

ASSOCIATED CONTENT Supporting Information Sequences of the CFPs targeting rotavirus and astrovirus, optimize the parameters of the CFPA system, the detection performance of CFPA when targeting both astrovirus cDNA and RNA samples, comparison between the present CFPA method and the classic isothermal amplification method (LAMP).

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Sequence-Specific Probe-Mediated Isothermal Amplification for the

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