Application of loop-mediated isothermal amplification (LAMP)-based ...

Protoplasma (2012) 249:417–422 DOI 10.1007/s00709-011-0293-2

ORIGINAL ARTICLE

Application of loop-mediated isothermal amplification (LAMP)-based technology for authentication of Catharanthus roseus (L.) G. Don Anis Ahmad Chaudhary & Hemant & Mohd Mohsin & Altaf Ahmad

Received: 4 March 2011 / Accepted: 22 May 2011 / Published online: 5 June 2011 # Springer-Verlag 2011

Abstract In this study, loop-mediated isothermal amplification (LAMP)-based molecular marker was developed for authentication of Catharanthus roseus, a medicinal plant. Samples of this plant were collected from different geographical locations in India. Random amplified polymorphic deoxyribonucleic acid (DNA) analysis of collected samples was carried out with 25 random primers. A 610-bp DNA fragment, common to all accessions, was eluted, cloned, and sequenced. Four LAMP primers were designed on the basis of sequence of 610 bp DNA fragment. LAMP reaction, containing 10× Bst DNA polymerase reaction buffer, Bst DNA polymerase, four inhouse designed primers, dNTPs, MgSO4, and betaine, was incubated at 65°C for 1 h. The resulting amplicon was visualized by adding SYBR Green I to the reaction tube. The data showed confirmatory results. Since the assay method is simple, sensitive, and cost-effective, it is a feasible method for identifying and authentication of C. roseus. Keywords Catharanthus roseus . RAPD . Loop-mediated isothermal amplification . Polymerase chain reaction

Introduction Medicinal plants serve as the primary therapeutic resource for most of the world’s population living in developing Handling Editor: Peter Nick A. A. Chaudhary : Hemant : M. Mohsin : A. Ahmad (*) Molecular Ecology Laboratory, Department of Botany, Faculty of Science, Hamdard University, New Delhi 110062, India e-mail: [email protected] A. Ahmad e-mail: [email protected]

countries (Koehn and Carter 2005; Jones et al. 2006). At the same time, the use of herbal preparations for healthcare purposes is gaining popularity in developed countries (Ernst 2005). The increased demand for botanical products is met by an expanding industry and accompanied by calls for assurance of quality, efficacy, and safety (Ernst 2006). Unequivocal identification and authentication of the plants used for production is an elementary and critical step at the beginning of an extensive quality assurance process. Unfortunately, substitution or adulteration either intentionally, e.g., motivated by the desire to maximize financial gains, or unintentionally, e.g., by clerical errors or lack of knowledge, are not rare occurrences and can have tragic consequences (Zhao et al. 2006). Identification of plants at the species level is traditionally achieved by careful examination of the specimen’s macroscopic and microscopic morphology. This work usually needs to be performed by a specially trained expert. However, morphological identification is often not possible when the original plant material has been processed. Therefore, additional methods of identification at the species level have been sought, and genome-based methods have been developed for the identification of medicinal plants starting in the early 1990s (Mizukami et al. 1993). Some of these molecular techniques have already been successfully applied to the classification and identification of herb plants. Among them, random amplified polymorphic DNA (RAPD; Fabbri et al. 1995; Belaj et al. 2001), amplified fragment length polymorphism (Angiolillo et al. 1999), and simple sequence repeat (Rallo et al. 2000; Sefc et al. 2000; Carriero et al. 2002; Cipriani et al. 2002) have been widely applied. Here, we have developed loop-mediated isothermal amplification (LAMP)-based method for identification of Catharanthus roseus L. Don. This method employs a deoxyribonucleic acid (DNA) polymerase and a set of four

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specially designed primers that recognize a total of six distinct sequences on the target DNA. An inner primer containing sequences of the sense and antisense strands of the target DNA initiates LAMP. The following strand displacement DNA synthesis primed by an outer primer releases a single-stranded DNA. This serves as template for DNA synthesis primed by the second inner and outer primers that hybridize to the other end of the target, which produces a stem–loop DNA structure. In subsequent LAMP cycling one inner primer hybridizes to the loop on the product and initiates displacement DNA synthesis, yielding the original stem–loop DNA and a new stem–loop DNA with a stem twice as long. The cycling reaction continues with accumulation of 109 copies of target in less than an hour. The final products are stem–loop DNAs with several inverted repeats of the target and cauliflower-like structures with multiple loops formed by annealing between alternately inverted repeats of the target in the same strand. Because LAMP recognizes the target by six distinct sequences initially and by four distinct sequences afterwards, it is expected to amplify the target sequence with high selectivity (Notomi et al. 2000). Since, this method is much simpler and more convenient assay that relies on visual detection, this method can be used for the detection of single herb species from a mixture through visible signals. Furthermore, no special apparatus is needed, which makes it more economical and practical than regular polymerase chain reaction (PCR) or real-time PCR. C. roseus (L.) G. Don (2n=16), commonly known as Madagascar periwinkle, is a member of the Apocynaceae family. This species is of great economic importance and is known because of its diverse medicinal properties. It has recently attracted the attention of researchers, especially for the studies of secondary metabolites and molecular biology investigations. It is a rich source of more than 125 mono-terpenoid indole alkaloids (Mishra and Kumar 2000) including the anticancer alkaloids (vincristine, vinblastine) and the antihypertensive alkaloid (ajmalicine). Other alkaloids such as serpentine have been reported to have a role in the treatment of diabetes and cardiovascular diseases (Mishra and Kumar 2000).

A.A. Chaudhary et al. Table 1 List of Catharanthus roseus (L.) G. Don Code

Accessions

Source

A1 A2

IC 49,595 EC 415,024

NBPGR, New Delhi NBPGR, New Delhi

A3

EC 49,580

NBPGR, New Delhi

A4 A5

IC 210,607 EC 120,837

NBPGR, New Delhi NBPGR, New Delhi

A6

IC 49,581

NBPGR, New Delhi

DNA isolation DNA was isolated from fresh or frozen leaves using a modified CTAB method (cetyl trimethyl ammonium bromide, Doyle and Doyle 1990). Briefly, leaf samples (0.2–0.5 g) were ground to fine powder in liquid nitrogen and transferred to a microcentrifuge tube containing freshly prepared equal volume of extraction buffer (100 mmol l−1 Tris buffer, pH 8.0, 20 mmol l−1 Na2EDTA, 1.4 mol l−1 NaCl, 2% CTAB,1% polyvinyl pyrrolidone). The suspension was gently mixed and incubated at 60°C for 60 min with occasional mixing. The suspension was then cooled to room temperature and an equal volume of chloroform : isoamyl alcohol (24:1) was added. The mixture was centrifuged at 13,000×g for 10 min. The clear upper aqueous phase was then transferred to a new tube containing 0.5 ml ice-cooled isopropanol and incubated at −20°C for 30 min. The nucleic acid was collected by centrifuging at 13,000×g for 10 min. The resulting pellet was washed twice with 70% ethanol containing 10 mmol l−1 ammonium acetate. The pellet was air-dried under a sterile laminar hood and the nucleic acid was dissolved in TE (10 mmol l−1 Tris buffer, pH 8.0, 1 mmol l−1 Na2EDTA) at 4°C. The contaminating RNA was eliminated by treating the sample with RNase A (20 μg μl−1) for 30 min at 37°C. DNA concentration and purity were determined by measuring the absorbance of diluted DNA solution at 260 nm and 280 nm. The quality of the DNA was determined using agarose gel electrophoresis stained with ethidium bromide. RAPD amplification

Materials and methods Plant materials Authentic seeds of C. roseus were procured from different parts of India through National Bureau of Plant Genetic Resources, New Delhi, India (Table 1). Seeds were grown in the Herbal Garden of Hamdard University, New Delhi, India. Fresh leaf samples were collected, frozen in liquid nitrogen and stored at −80°C until used for DNA isolation.

The RAPD amplification was performed according to the method developed by McClelland et al. (1995). PCR reactions were carried out in 25 μl reaction tubes using 25 random decanucleotide primers, OPAA-1, OPAA-2, OPAA-3, OPAA4, OPAA-5, OPAA-6, OPAA-7, OPAA-8, OPAA-9, OPAA-10, OPAA-11, OPAA-12, OPAA-13, OPAA-14, OPAA-15, OPAA-16, OPAA-17, OPAA-18, OPAA-19, OPAA-20 (Operon Technologies, USA), Bg26, Bg27, Bg28, Bg29 and Bg30 (Banglore Genei, India) (Table 2). Each reaction tube contained 50 ng template DNA, 1.5 mmol l−1 MgCl,

Application of loop-mediated isothermal amplification Table 2 Nucleotide sequences of selected primers with the number of amplified products and fragment sizes

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Primer code

Sequence 5′–3′

No. of amplified fragments

Fragment size (bp)

OPAA-01 OPAA-02

AGACGGCTCC GAGACCAGAC

55 45

900–113 969–189

OPAA-03

TTAGCGCCCC

42

1,068–109

OPAA-04 OPAA-05

AGGACTGCTC GGCTTTAGCC

60 38

988–100 977–135

OPAA-06

TCAAGCTAAC

92

1,210–180

OPAA-07 OPAA-08

CTACGCTCAC TCCGCAGTAG

39 76

10,114–116 938–106

OPAA-09

AGATGGGCAG

48

947–105

OPAA-10 OPAA-11

TGGTCGGGTG ACCCGACCTG

68 38

1,154–153 995–175

OPAA-12

GGACCTCTTG

40

885–128

OPAA-13 OPAA-14

GAGCGTCGCT AACGGGCCAA

53 47

954–225 964–328

OPAA-15

ACGGAAGCCC

70

998–115

OPAA-16 OPAA-17

GGAACCCACA GAGCCCGACT

80 77

1,010–141 998–116

OPAA-18 OPAA-19

TGGTCCAGCC TGAGGCGTGT

57 29

895–281 997–135

OPAA-20 Bg26 Bg27

TTGCCTTCGG AAGCCTCGTC TGCGTGCTTG

Nil 112 87

975–186 1,163–313

Bg28 Bg29 Bg30

GACGGATCAG CACACTCCAG TGAGTGGGTG

25 Nil 13

Total

570–283 476–383

1,291

300 μmol l−1 of dNTPs, 1× Taq DNA polymerase buffer, 25 pmol decanucleotide primer and 2 units of Taq DNA polymerase (Promega, USA). Amplification was performed in a DNA thermal cycler (Ependroff, USA), using the following conditions : 95°C for 3 min; 40 cycles at 94°C for 30 s, 50°C for 1 min and 72°C for 1 min; final extension at 72°C for 5 min. PCR products were resolved on 1.2% agarose gel in 1× TAE buffer, agarose gel containing 0.5 μg ml−1 ethidium bromide visualized under ultraviolet (UV) light and photographed using gel documentation system (Image Master VDS, Pharmacia, USA). RAPD markers suffer from a lack of reproducibility. Consequently, to confirm the electrophoretic patterns and the obtained polymorphic bands, every PCR was repeated twice under the same conditions of composition of reaction volume, amplification profile, and thermalcycler.

pGEM®-T easy vector (Promega) following the manufacturer’s instruction. The ligated plasmid was introduced into Escherichia coli strain DH5α, following the protocols for preparing competent cells and transformation using the calcium chloride method (Sambrook and Russell 2001). White colonies were picked from LB-X-gal plates and grown overnight in LB medium containing ampicilin. The plasmid DNA was isolated from the bacterial culture using plasmid isolation kit (QIAGEN). The inserted fragment was sequenced at the Center for Genomic Application, New Delhi, India, with T7 primer. Nucleotide sequence of 610 bp was identical for all the six accessions (Fig. 1). This sequence was used for designing primers for LAMP reaction.

Cloning and sequencing of specific RAPD fragment

Four oligonucleotide primers, forward inner primer (FIP), back inner primer (BIP), and two outer primers (F3 and B3), were designed by using the sequence of DNA from 610bp RAPD amplicon. All primer sequences were designed with the software program Primer Explorer

A band of 610-bp, which is common in all the six accessions, was excised from gel and eluted using a Gel Extraction Kit (QIAGEN, Germany). The eluted DNA was cloned into

Primers design for LAMP

420 Fig. 1 a Nucleotide sequence of RAPD amplicon (610 bp) of C. roseus and underlines indicate the designed LAMP primers. b LAMP primers

A.A. Chaudhary et al.

A 0

5 - accgggttgc taacatgagc gaagcctggt gaccccgatt cgtgttgcat cttatcctag gcgggggggg

71

ctatttgtga

gattattgga

aggtatgaaa

141

tctaacggtg ccgctacacg attcaggctt atggtccact cccaattgga ctcaccataa aattcttaaa

211

ccactgtaac taaaacgggt caagatatgg gttttgctaa cttctcaata aaataccata acgaatattt

281

ttgttaatta

351

atactttgtt gtgatcctca gctcaatatt gaggctaggg accgttttga tgccacctgc atggaacggt

421

agagaagttg atgcataatt tgcagatagc ttccaagaag ctaacatgga taccccgggc gtgtatgacg

491

gactgtgggc taagtgggat gacctgaagt attaggccat gtgtgagcag acctaaagaa aaaggggcga

tcaaaaaaat

atcttttttc

aactgtgcct

catttttttt

tctttttttg

tgttgacttt

ttgcttttaa

taacttgttt

ttttgtagag

tatcggagaa

F2

F3 F1c

B1c B2

B3

561 tggacgatgc ttaaaggacg ncgggggctt gggaagcgcc gtataccgg - 3

B LAMP Primers

Outer forward and backward Primers

F3 b3

5 -ctttgttgtgatcctcagc - 3 5 -gcccctttttctttaggtc - 3

Bases 19 19

Inner forward and backward Primers FIP (F1c+F2) BIP (B1+B2c)

5 -ctgcaaattatgcatcaacttctct-atattgaggctagggaccg- 3 5 -atagcttccaagaagctaacatgg-tggcctaatacttcaggtc- 3

44 43

V3 (http://primerexplorer.jp/elamp3.0.0/index.html). The primers were selected based on the criteria described by Notomi et al. (2000). Briefly, the design of the two outer primers, F3 and B3, is the same as that of regular PCR primers, while the design of the two inner primers, FIP and BIP, is different from that of PCR (Notomi et al. 2000). FIP consists of the sense sequence of F2 at the 3′ end and the F1c region at the 5′ end that is complementary to the F1 region. BIP consists of a B2 region at the 3′ end that is complementary to the B2c region and the same sequence as the B1c region at the 5′ end (Fig. 1). LAMP assay LAMP reaction (25 μl) contained the one pair of outer primers (0.2 μM) and one pair of inner primers (1.6 μM), 2.5 μl of 10× Bst DNA polymerase reaction buffer [1 μl containing 20 mM Tris–HCl, 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100 (pH 8.8)], 400 μM each dNTP, 1 μl of an 8 Uμl−1 concentration of Bst DNA polymerase (New England Biolabs, MA), 2 mM MgSO4 (2 μl), 5 μl of betaine (Sigma-Aldrich, St. Louis, MO, USA), and 5 μl of doublestranded target DNA. The LAMP reaction was performed in a heating block (Genei, India) at 65°C for 1 h. For comparison, the reaction was also performed by using a conventional thermal cycler (Bio-Rad, USA).

Fig. 2 RAPD electrophoresis profile of six accessions of Catharanthus roseus amplified with OPAA-10 primer. Lanes 1–6 correspond to the six accessions listed in Table 1. Lane M molecular marker 100 bp (inno train, Germany). The numbers on the left of the figure indicate the DNA size markers in bp

Application of loop-mediated isothermal amplification

421

Fig. 3 Analysis of LAMP result under UV transilluminator. A1–A6 shows accessions of C. roseus. C Control

Visualization of LAMP product The inspection for amplification was performed through observation of a color change following addition of 1 μl (1:1,000) of SYBR Green I dye to the tube. This color was visualized by naked eye without any UV source and produced florescence in under UV transilluminator.

Results RAPD method was performed in search for DNA polymorphisms, which can be used for generating informative LAMP-based molecular markers and defining the individual cultivars. Twenty five RAPD primers (see “Materials and methods”) were used. Only the fragments confirmed by repeated amplifications were considered useful for generating LAMP markers. Preliminary screening of 25 random decanucleotide primers showed that 23 primers were able to prime genomic DNA of C. roseus and resulted in amplified PCR products of a variable number of DNA bands (13–112 bands primer−1). A total of 1,291 DNA bands were obtained. Amplification of C. roseus species with OPAA-6, OPAA-10, OPAA-17, and OPAA-19 produced good quality, reproducible fingerprint patterns and showed a high level of consistency of fingerprints among samples of the same species collected from different localities (Fig. 2). Several specific RAPD fragments of high intensity and reproducibility were eluted, cloned, and Fig. 4 Analysis of LAMP result on a naked eye without using any UV source. A1–A6 shows accessions of C. roseus. NC Negative control

sequenced. Nucleotide sequence of 610 bp amplicon, identical for all the six accessions of C. roseus, was used for designing primers for LAMP reaction. The LAMP reaction relies mainly on autocycling strand displacement DNA synthesis that is similar to the cascade rolling-circle amplification reported by Hafner et al. (2001). The minimum LAMP reaction unit consists of two outer primers (F3 and B3), two inner primers (FIP and BIP), and target DNA. Each inner primer contains two distinct sequences corresponding to the sense and antisense sequences of the target DNA and form stem–loop structures at both ends of the minimum LAMP reaction unit. These stem–loop structures initiate self-primed DNA synthesis and serve as the starting material for subsequent LAMP cycling reaction. The LAMP products were visualized on a UV transilluminator at 302 nm (Fig. 3) and on a naked eye without using any UV source (Fig. 4).

Discussion C. roseus has been studied extensively in India because of its widespread used in herbal medicine. We have developed a credible and convenient LAMP-based method for the identification of C. roseus. The LAMP operation is quite simple; it starts with the mixing of buffer, primers, DNA, and DNA polymerase in a tube, and then the mixture is incubated at 65°C for a certain period. For the visualization of product, SYBR Green I was added. The tubes can also be

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inspected for white turbidity with the naked eye after a pulse spin to deposit the precipitate in the bottom of the tube (Mori et al. 2001). However, detecting a small amount of the white precipitate by the naked eye is not always easy; therefore, the detection limit is apparently inferior to that of electrophoresis. To increase the rate of recognition by the naked eye, addition of SYBR Green I to the reaction solution is convenient (Hill et al. 2008). LAMP amplification is rapid (results can be obtained in less than 1 h), easy to perform, and low in cost. Because of to its easy operation without any sophisticated equipment, it will be simple enough for use in small-scale industries, hospitals, and testing laboratories in developing countries.

Conflicts of Interest None

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