A novel method of probe-melting curve for nucleic aciddetection without occupying f uorescence channel

Yng Hn, Shngzhi Ji, Xingzhong Ye, Yimin Li, Yixun Zhng＊

aThe College of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University,

Shenyang 110016, China;

bDepartment of research and development, Beijing Wantai Biological Pharmacy Enterprise

Co., Ltd, Beijing 102206, China

Regular article

A novel method of probe-melting curve for nucleic acid
detection without occupying f uorescence channel

Yang Hana, Shangzhi Jib, Xiangzhong Yeb, Yimin Lib, Yixuan Zhanga＊

aThe College of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University,

Shenyang 110016, China;

bDepartment of research and development, Beijing Wantai Biological Pharmacy Enterprise

Co., Ltd, Beijing 102206, China

A novel method, probe-melting curve method, has been established to increase the detection throughputs within one f uorescence channel. Besides using an asymmetric PCR assay, this method employs another specially designed probe with a 5′ terminus modif cation. The method has been demonstrated by detection of rotavirus as model system. The sensitivity of probe-melting curve method was proved to be equal to the conventional TaqMan real-time RT-PCR. And the experiment results were cross checked by agarose gel electrophoresis. The successful establishment of probe-melting curve method breaks numbers of f uorescence channels from the restrictions and enlarges the application f elds of current real-time PCR instruments.

Real-time PCR; RT-PCR; probe-based method; melting curve method

1 Introduction

Real-time fluorescence quantitative PCR as a sensitive, specific, high automation and low contamination method has been one of the most rapid and accurate tools in all life science f elds [1]. In recent years, real-time PCR research in the pharmacology and identification of traditional Chinese medicine has also been great progress. Traditional Chinese medicine treatment has a strong overall concept, and the information it obtained is always ambiguous, subjective and uncertain at the same time. With the application of the real-time PCR technology, the pharmacological eff cacies and mechanisms of traditional Chinese medicine can be understood at a molecular level. The mRNA of some key factors in body can be quantif ed which can be a monitoring index of medicine effect and can be used to evaluate the curative effect of traditional Chinese medicine. Real-time PCR technology as an effective means can also be applied for identification and quality control of traditional Chinese medicine [2-4]. Currently, there are two main methods of Real-time f uorescence PCR: one is based on DNA f uorescent dyes, such as SYBR Green I. This kind of f uorescent dye can be embedded into the groove structure of DNA, and the fluorescent of dye will be enhanced more than 1000 times when it is combined withDNA. So, real-time quantitative PCR with SYBR Green I is a kind of convenient and effective method to detect and identify variedly pathogens [5, 6]. However, it had no specific to dsDNA, including primer dimmer, and the sensitivity of dye-based methods is usually low which limits its application. The other is probe-based method which combines fluorescence resonance energy transfer (FRET) with conventional PCR and using a targetspecific sequence named probe. The fluorescence released only when the probe hybridizes with target template, so the probe-based method has high specificity and sensitivity than dye-based method. The probe-based method mainly includes hydrolysis probes, molecular beacons probe, FRET probes and so on. TaqMan probe is one kind of hydrolysis probes which will be hydrolyzed by 5′to 3′ exonuclease activity of DNA polymerase and emits fluorescence when it hybridizes with target template, and it is the most widely used probe. Besides, be labeled with different kinds of fluorophore, different kinds of samples can be multiplex detected by TaqMan probes [7]. Moreover, the probe-based methods can also be used to detect mutation of strains with melting curve analysis [8].

The probe-based method greatly speeds up the process of Real-time f uorescence quantitative PCR. However, real-time fluorescence quantitative PCR instruments in the current market have limited detecting channels, and the throughputs of probebased method are always difficult to increase. What’s more, the manufacturers of PCR instruments often chose one channel for f uorescence calibration, and another of the channels is always occupied by internal control when test samples. These have greatly reduced the throughputs of multiplex PCR. In addition, the effect of each f uorescence channel is slightly different. Therefore, the development of multiplex real-time fluorescence quantitative PCR is greatly limited by the current fluorescence quantitative PCR instrument.

In order to break through the restriction of current fluorescence quantitative PCR instrument, and enlarge its application fields, this work has set up a novel method based on probe-melting curve analysis which can test pathogens without occupying any fluorescence channel. More importantly, this method has the same sensitivity with the conventional TaqMan real-time RT-PCR. This method has important value, and it provides new idea for development of real-time f uorescence quantitative PCR in the future.

2 Equipments and materials

Bio-Rad CFX-96 Real-time PCR Detection System (Bio-Rad Laboratories Co., Ltd); Electrophoresis Apparatus (JY1600C, Beijing Liuyi Instrument Factory); Ultraviolet Detector (WFH-201B, Shanghai Jingke Industrial Co., Ltd).

Cell cultures of Rotavirus (Provided by Beijing Wantai Biological Pharmacy Enterprise Co., Ltd); RNA/DNA extraction kit (GenMagBio, Beijing, China); RT-PCR Quick Master Mix (Toyobo Life Science Department, Shanghai); Mn(AcO)2(Sigma-Aldrich Corporation); TE (10mM Tris, 1mM EDTA, pH 8.0); TAE (40 mM Tris-acetate 1 mM EDTA, pH 8.0).

3 Methods

3.1 Nucleic acid extraction

Rotavirus was provided by Beijing Wantai Biological Pharmacy Enterprise Co., Ltd. The virus genomic RNA was extracted from 200μL of culture with RNA/DNA extraction kit (GenMagBio, Beijing, China) according to its manufacturer’s protocol. And the rotavirus RNA was then frozen and stored at–80°C.

3.2 Primer and probe design

On the basis of the known gene sequences of rotavirus strains in GenBank, the sequence data were analyzed by DNAStar Software 5.0 to find out the proper conserved regions for primers design. And then, the primers and probes of rotavirus were designed by Primer Premier 5.0, and the melting temperature, self-dimer and heterodimer properties of the primers and probes were examined in silico using Oligo Analyzer 7.0.

The primers and probes of rotavirus used were as follows: R-F (5′-TGTTGCTCAAGATGGAG TCTACTCAG-3′), R-R (5′-CCATTAATTCTAATGT TGAAGTGGCA-3′), the conventional TaqMan probe for control: R-P (5′-FAM-AGATGGTAAGC TCCATTATTAACACTTCTT-MGB-3′), the probe for melting curve analysis: R-MP (5′-FAM-NHTGGTAAGCTCCATTATTAACACT-Eclipse-3′). The R-F primer and the two probes were designed by the same chain. The size of the amplicon is 102 bp. The GenBank serial number of the reference rotavirus strain in this assay is FJ423119.1. All primers and probes were synthesized by Takara Biotechnology (Dalian) Co., Ltd.

3.3 Optimization ratio of primers of probe-melting curve assay

In order to screen out the best asymmetric PCR condition, the ratio of forward primers to reverse primers were set to 1:1, 1:5, 1:10, 1:25. The reaction was performed in a total volume of 50μL, containing 25μL RT-PCR Quick Master Mix, 2.5 mM Mn(AcO)2, 0.3μM R-F, corresponding proportion amounts of R-R, 0.2μM R-MP and 20μL of rotavirus RNA sample.

3.4 Conventional TaqMan real-time PCR

The reaction was performed in a total volume of 50μL, containing 25μL RT-PCR Quick Master Mix, 2.5 mM Mn(AcO)2, 0.3μM R-F, 0.3μM R-R, 0.1μM R-P and 20μL of rotavirus RNA sample.

3.5 The reaction protocols

The reaction cycling parameters consist of denaturation for 1 min at 95°C; 20 min reverse transcription at 61°C (for reverse transcription); followed by 5 cycles touch-down PCR of 95°C for 15 s and 64°C for 30 s by decrement the temperature of –1°C of each cycle; followed by 40 cycles of 95°C for 15 s, 62°C for 30 s. Fluorescence readings were taken during the anneal/extending step (62°C incubation). Followed by a melting analysis procedure consisted of incremental temperature from 40°C to 95°C at a rate of 0.5°C per second.

3.6 Sensitivity of probe-melting curve method

Ten-fold serial dilutions of rotavirus RNA sample with TE buffer from E6 to E1 (E6, E5, E4, E3, E2, E1) were assayed separately by probemelting curve method and conventional TaqMan real-time PCR to determine their sensitivity. Water contrast group was assayed for negative control at the same time.

3.7 Agarose gel electrophoresis

10μL of amplified products were loaded in a 3% agarose gel in TAE (40 mM Tris-acetate 1 mM EDTA, pH 8.0) buffer for electrophoretic analysis at 110 V for 40 min with ethidium bromide staining. After the electrophoretic, the agarose gel was visualized through Ultraviolet Detector (WFH-201B, Shanghai Jingke Industrial Co., Ltd).

4 Results

4.1 The proper ratio of primers of probe-melting curve assay

The ratio of forward primers to reverse primerswere set to 1:1, 1:5, 1:10, 1:25 to screen out the best asymmetric PCR condition. From figure 1, there is no plot in amplif cation phase, and the ratio 1:5 showed the best result in melting curve phase, followed by 1:10, 1:25. But the ratio of 1:1 did not show very obvious peak. The Tms of the positive results all at about 54°C which is consistent with the values calculated in silico (53.8°C).

4.2 Sensitivity of probe-melting curve method

The sensitivity of both detection methods, probe-melting curve method and conventional TaqMan real-time PCR, were compared by the detection of ten-fold serial dilutions of rotavirus RNA samples. The detection limits of the two methods were both the concentration of E2 (Figure 2). And this indicated that probe-melting curve method had the same sensitivity with conventional TaqMan real-time PCR.

4.3 Agarose gel electrophoresis

As shown in Figure 3, the results of electrophoresis were complied with the product sizes: 102bp, indicating the rotavirus RNA was amplified effectively, and this validated the feasibility of probe-melting curve method. Meanwhile, there is no more bandings in lanes suggested good design of primers and probes.

5 Discussions

It is the inevitable trend of development that most infectious diseases will be tested by multiplex real-time PCR in the future. Because conventional test methods of infectious diseases still have their detecting limits, such as low-throughput, timewasting, reagent-wasting and low sensitivity, the multiplex real-time PCR is an ideal solution for infectious diseases detection due to its simple, rapid, convenient and efficient. The strictly screening of HBV, HCV and HIV in the blood, the coxsackie A16 (CA16) and enterovirus type 71 (EV71) detection of hand, foot and mouth disease, and the rotavirus, norovirus and enteric adenoviruses monitoring of the intestinal diarrhea virus [9] all urgently need the application of multiplex real-time PCR. While molecular beacons, duplex scorpionprimer and FRET probes [10] have greatly developed the detection capabilities and applications of real-time PCR, they are limited in multiplex real-time PCR due to their complex design and high cost.

The probe-melting curve assay is a novel method which breaks out of channels and can be applied in most real-time PCR instruments. This approach is intended to detect samples with a special design probe in melting phase on the basis of without occupy the amplification phase. And the meltingpeak value has a positive correlation with amounts of PCR products so as to get the information of qualitative analysis and semi-quantitative analysis.

To achieve those effects above, the probe used in probe-melting method was modified with chemical groups at the 5′ end f rstly. And this kind of modification could avoid the probe from being cut by DNA polymerase in the process of amplif cation. In this assay, the probe was modified with amino group, and it could be modified with sulfhydryl group or biotin too. But in practice, some of the probes would be cut in extension if they combined with template firmly [11]. In addition, if the probe was too long, it would have some signif cant change of fluorescence in amplification phase even if the probe had not be cut [12]. This phenomenon could be a result of the linear structure of the probe. That meant the excessively long linear structure might lead to a high background of fluorescence. When the probe was in a free state, the fluorescent group was relatively close to the quencher group while the probe would be straightened and the distance between f uorescent group and the quencher group got longer when it hybridized with template and these would release f uorescent signal according to FRET. Therefore, based on modifying the probe, this method lowered the Tm value of the probe by shortening its length, and these would make the probe not hybridize with template during PCR so as to solve the problem of generating signals in amplification phase. As shown in the Fig (A) and (B), there was no signal detected in the whole amplification process indicating a proper design of probe in this assay.

While at the initial stage of melting, the probe was combined with template and it was straightened. The distance between fluorescent group and the quencher group was relatively long at this time. As the temperature rose, the probe and the template were separated gradually and the distance between f uorescent group and the quencher group got shorter when the probe recovered to the free state. The fluorescent signal reduced which showed a downward trend in fluorescence intensity and a positive peak in melting curve analysis. The point which f uorescent signal decreased most quickly was the Tm value of the probe, and it corresponded to abscissa value of melting curve peak value. Inthis assay, the Tms of all positive results all were about 54°C which accorded with theoretical values 53.8°C. In addition, to obtain enough single-stranded DNA and enhance the f uorescent signal in melting, it was necessary to apply the asymmetric PCR in this method [13]. This was further conf rmed by the experimental results. From f gure 1 (B), the primers ratio of 1:1 did not show a good melting peak while the ratio of 1:5 had an obvious result.

The probe-melting curve method showed the same sensitivity with the conventional TaqMan realtime RT-PCR in this assay. In addition, the probe used in this method had no strict structure and had no requirement of the distance to the primers. These advantages greatly benefit the flexibility of probe design. Most importantly, this method can test sample without occupying any f uorescence channel which means the fluorescence channel can be used to detect extra sample in amplification phase by conventional TaqMan real-time PCR.

In conclusion, the probe-melting curve method features a high sensitivity, simple design of probe, and an excellent compatibility as well. The successful establishment of probe-melting curve method breaks numbers of fluorescence channels from the restrictions and has broad prospects in application in the future.

Acknowledgement

This work was supported by Shenyang Pharmaceutical University, Beijing Wantai Biological Pharmacy Enterprise Co., Ltd.

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* Author to whom correspondence should be addressed. Address: School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang 110016, China; Tel.: +86-24-23986576; Email: zhangyxzsh@163.com

Received: 2014-03-05 Accepted: 2015-07-12