WO2016064264A1

WO2016064264A1 - Multiplex pcr kit for the detection of pig, dog, cat, rat and monkey derived materials for halal authentication

Info

Publication number: WO2016064264A1
Application number: PCT/MY2015/000085
Authority: WO
Inventors: Ali EAQUB; Razzak ABDUR; O.A. Abd Hamid SHARIFAH BEE BINTI
Original assignee: University Of Malaya
Priority date: 2014-10-23
Filing date: 2015-10-21
Publication date: 2016-04-28

Abstract

The present invention discloses a multiplex polymerase chain reaction (Multiplex PCR) kit for multiple target detection in a single assay platform for Halal authentication. The Multiplex PCR kit disclosed according to the present invention comprises of five sets of forward and reverse specifically designed primers for DNA amplification to identify five meat species forbidden in Islamic foods such as pig, dog, cat, rat and monkey. In another embodiment, the multiplex PCR kit according to the present invention comprising five different sets of species-specific primers that specifically detect five different segments (108 to 172 bp) of three different mitochondrial genes of the said five species.

Description

MULTIPLEX PCR KIT FOR THE DETECTION OF PIG, DOG, CAT, RAT AND MONKEY DERIVED MATERIALS FOR HALAL AUTHENTICATION

INCORPORATION OF SEQUENCE LISTING

A computer readable form of the sequence containing the file named "PIC_UM768.txt", which is 4,096 bytes in size on disk (as measured in Microsoft Windows 8) are provided herein and are herein incorporated by reference. The Sequence Listing consists of SEQ ID NO: 1 to SEQ ID NO: 10.

FIELD OF THE INVENTION

The present invention relates to multiple target detection in a single assay platform for Halal authentication. In more specific, the present invention further relates to five sets of specifically designed primers for multiplex polymerase chain reaction (Multiplex PCR) kit to identify five meat species forbidden in Islamic foods, i.e. pig, dog, cat, rat and monkey.

BACKGROUND OF THE INVENTION

Halal is an Arabic word which means permitted. This refers to the acceptance in Islamic religion or law (Sahilah et al., 2011). Animals are categorized into two groups, namely, halal and haram animals based on Islamic law. To be shariah compliants, halal animals such as chicken, cow, goat, sheep and etc, have to be slaughtered appropriately according to the Syariah Law. On the other hand, haram animals such as pig and its inheritance or any other derived food, dog, rat are not halal and cannot be made them halal even they are slaughtered according to the Islamic Law. Apart from that, there are also group of halal animals, such as fish and crustaceans which do not need to be slaughtered to be halal (Sahilah et al., 2011).

Thus, meat species identification as well as Halal authentication is a main concern in most countries such as Asia, France, Russia, Sweden, Germany, Switzerland, Greece, Spain, Italy, United Kingdom, South and North America and etc. (Chandrika et al., 2009). In another aspect, authentication of declared components in foods is an ever increasing public demand and also a key priority in policy making and regulatory bodies since it is necessary for the safeguard of public health, consumers' lifestyle, food choice, religious faith, fair-trade economy and wildlife in natural. The turnover of halal food items has crossed USD2.1 trillion and is expanding rapidly even among the non-Muslim consumers because of its perceived quality attributes and significantly reduced risk to be a carrier of zoonotic diseases. It has been believed that the causative agent of one of the most fatal human disease acquired immune deficiency syndrome was transmitted to human race from African Chimpanzee which is forbidden for consumption in Islam. Perceiving the huge opportunities of the halal food markets, even the European food industries are investing in the production of halal foods. Technologically species-specific PCR seems to be the best and is considered as a robust method in comparison with other methods such as single nucleotide polymorphism (SNP) analysis, PCR-RFLP, PCR-RAPD and DNA bar coding.

Although, several multiplex PCR assays have been documented for the identification of various animal species (Table 1), none of them has been aimed at the authentication of prohibited species in Islamic foods. In view of foregoing, the present inventor has developed a multiplex Species-specific PCR assay kit for the identification of five meat species forbidden in Islamic Shariah complaint hygienic foods.

Species-specific PCR assays using specifically designed primers under restrictive PCR conditions is conclusive in species identification and eliminates the need of restriction digestion and/or sequencing of PCR products. However, compared to conventional single-species PCR systems, multiplex PCR is greatly promising since it offers multiple target detection in a single assay platform reducing both cost and time. Table 1

Identified Species Target Product size (bp) Detection Reference

Gene(s) limit (ng)

Cattle, Pork, Chicken, cyt b 274, 398, 227, 157, 0.25 (Matsunaga, Goat, Sheep, Horse 331, and 439 et al., 1999)

Ruminant (Bos taurus, 12S and 16S 104 - 106, 183, 220 0.0025- (Dalmasso, Capra hircus, Ovis aries) rRNA and - 230 and 290 0.025 et al., 2004) Poultry, Fish and Pork tRNA-Val

Horse and Pig cyt b 439 and 398 0.25 (Di Pinto, et al., 2005)

Cattle, sheep, pig and 16S rRNA 271, 274, 149, and 0.1-0.2 (LUO, et chicken 266 al., 2008)

Ruminant, poultry and 12S and 16S 104, 183, and 290 (Ghowati porcine rRNA et al., 2009)

Yak and Cattle mt 12S 290 (Yak), 290 and 0.5 (Yin et al., rRNA 159 (Cattle) 2009)

Bovine, Poultry, Ovine tRNA-Val 124, 183, 225 and 0.5- 5 (Zha et al., and Porcine and 290 2010)

Wapiti, Sika deer, Tarim D-loop and 141, 230, 246, 272 0.02- 0.5 (Zha et al., red deer, Red deer, 16S rDNA and 307 2011) Reindeer

Red deer, Sika deer, D-loop 199, 299, 245 and 0.05-1 (Eung Soo, Wapiti and Reindeer 375 2012)

Chicken, beef, mutton, cyt b 216, 263, 322, and 0.001 (Zhang, pork 387 2013)

Pork, lamb, chicken, cyt b, t-Glu- 100, 119, 133, 155, 7-21 fg (Kitpipit et ostrich, horse and beef cyt b, COI, 253, 311 al., 2014)

12S rRNA SUMMARY

An object of the present invention is to provide a multiplex PCR kit for the detection of five meat species forbidden in Islamic foods. These five types of meat species are selected from the group consist of pig, dog, cat, rat and monkey.

In one embodiment according to the method of the present invention, the multiplex PCR kit comprising five different sets of species-specific primers that specifically detect five different segments (108 to 172 bp) of three different mitochondrial genes of the said species. Mitochondrial gene-targets of these lengths are highly stable under extreme conditions of physical, chemicals and environmental stresses, suggesting the assay validity even under stressful conditions such as degraded specimens.

Another object of the present invention is to provide the use of a combination of 5 pair of primer sequences or 5 nucleotide fragment set comprising base sequence (SEQ ED NO: 1 ) to (SEQ ID NO: 10) represented by the following sequences (SEQ ID NO: 1) to (SEQ ED NO: 10) for DNA amplification of the five target species comprising the following nucleotide sequences fragments or the base sequences complementary thereto:

5'- -CCATCCCAATTATAATATCCAACTC-3 ' (SEQ ID NO: 1)

5'· -TGATT ATTTCTTGGCCTGTGTGT-3 ' (SEQ ID NO: 2)

5'- -TGGCTCTAGCCGTTCGATTA-3 ' (SEQ ID NO: 3)

5'- - AAGGC AAC AGC AAATTCTAGG-3 ' (SEQ ID NO: 4)

5'· -GGAATAATGTTTCG ACCACTAAGC-3 ' (SEQ ID NO: 5)

5'· -TGCCTGAGATGGGTATTAGG AT-3 ' (SEQ ID NO: 6)

5'- ■ ATC ATC AGAACGCCTT ATTAGC-3 ' (SEQ ID NO: 7)

5'- ■ AGGTTCGTCCTTTTGGTGTA-3 ' (SEQ ID NO: 8)

5'· GAGACCTCC AAC AAATACTAGC-3 ' (SEQ ID NO: 9)

5'- ■CTCTATGGC AGAAGGTAGTCAG-3 ' (SEQ ID NO: 10)

Yet another object of the present invention is to provide a method to detect pig, dog, cat, rat and monkey derived materials in a single assay platform, saving the cost and time of the separate PCR assays identifying the said species by at least five folds. The method according to the present invention comprising steps of applying species- specific primer design; conducting specificity and cross-specificity testing using simplex PCR; amplifying five multi-species DNA targets using multiplex PCR; analyzing amplified PCR products by Electrophoresis.

A further object of the present invention is to provide combination of five different sets of primers to detect cat, dog, pig, monkey and rat derived materials in Halal Foods. Each set of primers could also be independently used in separate identification of individual species.

The present invention is highly needed for a rapid, sensitive, cost-effective reliable approach for detection of pig, dog, cat, rat and monkey derived materials in a single assay platform.

A further object, features and advantages of the present invention will be readily apparent from the following description.

BRIEF DESCRIPTION OF THE DRAWING/FIGURES

The accompanying drawings, which are included to provide a further understanding of the present invention are incorporated in and constitute a part of this specification, illustrate embodiments of the present invention and together with the description serve to explain the principles of the present invention. Figure 1 shows a gel image for the development of multiplex PCR;

Figure 2 shows a corresponding electropherogram of lane 1 illustrates the peaks for negative control with amplicon size of 1500 bp and 15 bp; Figure 3 shows a corresponding electropherogram of lane 2 illustrates the peaks for duplex PCR of dog (amplicon size 163 bp) and pig (amplicon size 141 bp).; Figure 4 shows a corresponding electropherograms of lane 3 illustrates the peaks for triplex PCR of dog (amplicon size 163 bp), pig (amplicon size 141 bp) and rat (amplicon size 108 bp); Figure 5 shows a corresponding electropherograms of lane 4 illustrates the peaks for triplex PCR of cat (amplicon size 172 bp), pig (amplicon size 141 bp) and rat (amplicon size 108 bp);

Figure 6 shows a corresponding electropherograms of lane 5 illustrates the peaks for tetraplex PCR of cat (amplicon size 172 bp), dog (amplicon size 163 bp), monkey (amplicon size 129 bp) and rat (amplicon size 108 bp);

Figure 7 shows a corresponding electropherograms of lane 6 illustrates the peaks for multiplex PCR of cat (amplicon size 172 bp), dog (amplicon size 163 bp), pig (amplicon size 141 bp), monkey (amplicon size 129 bp) and rat (amplicon size 108 bp);

Figure 8 shows a corresponding electropherograms of lane 7 illustrates the peak for PCR of rat (amplicon size 108 bp);

Figure 9 shows a corresponding electropherograms of lane 8 illustrates the peak for PCR of monkey (amplicon size 129 bp);

Figure 10 shows a corresponding electropherograms of lane 9 illustrates the peak for PCR of pig (amplicon size 141 bp);

Figure 11 shows a corresponding electropherograms of lane 10 illustrates the peak for PCR of dog (amplicon size 163 bp);

Figure 12 shows a corresponding electropherogram of lane 11 illustrates the peaks for PCR of cat (amplicon size 172 bp); Figure 13 shows the gel image for the sensitivity of the Multiplex PCR;

Figure 14 shows a corresponding electropherograms of lane 1 illustrates a negative control of 0 ng DNA template of the said five meat species;

Figure 15 shows a corresponding electropherograms of lane 2 illustrates the sensitivity of the multiplex PCR with 10 ng DNA template of the said five meat species; Figure 16 shows a corresponding electropherograms of lane 3 illustrates the sensitivity of the multiplex PCR with 5 ng DNA template of the said five meat species;

Figure 17 shows a corresponding electropherograms of lane 4 illustrates the sensitivity of the multiplex PCR with 1 ng DNA template of the said five meat species;

Figure 18 shows a corresponding electropherograms of lane 5 illustrates the sensitivity of the multiplex PCR with 0.5 ng DNA template of the said five meat species; Figure 19 shows a corresponding electropherograms of lane 6 illustrates the sensitivity of the multiplex PCR with 0.2 ng DNA template of the said five meat species;

Figure 20 shows a corresponding electropherograms of lane 7 illustrates the sensitivity of the multiplex PCR with 0.1 ng DNA template of the said five meat species;

Figure 21 shows a corresponding electropherograms of lane 8 illustrates the sensitivity of the multiplex PCR with 0.05 ng DNA template of the said five meat species; Figure 22 shows a corresponding electropherograms of lane 9 illustrates the sensitivity of the multiplex PCR with 0.02 ng DNA template of the said five meat species; Figure 23 shows a corresponding electropherograms of lane 10 illustrates the sensitivity of the multiplex PCR with 0.01 ng DNA template of the said five meat species; and

Figure 24 shows a corresponding electropherograms of lane 11 illustrates a negative control of 0 ng DNA template of the said five meat species.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

In the following detailed description, reference is made to various specific embodiments in which the present invention may be practiced. These embodiments are described with sufficient details to enable those methods in the present invention to be practiced, and it is to be understood that other embodiments may be employed and that structural and logical changes may be made without departing from the scope of the present invention. In general, the present invention disclosed a multiplex PCR kit for the detection of pig, dog, cat, rat and monkey derived materials in food preparation. The multiplex PCR kit is able to detect pig, dog, cat, rat and monkey derived materials in a single assay platform. The present invention is able to save the cost and time of the separate identifying PCR assays of the said species by at least five folds.

In one embodiment, the multiplex PCR kit according to the present invention consists of five different sets of species-specific primers that specifically detect five different segments (108 to 172 bp) of three different mitochondrial genes of the said species. Mitochondrial gene-targets of these lengths are highly stable under extreme conditions of physical, chemicals and environmental stresses, suggesting the assay validity even under stressful conditions such as degraded specimens. In a specific embodiment, the five different sets of species-specific primers represented by the following sequences (SEQ ID NO: 1 to 10) according to the present invention comprising the following nucleotide fragments or the base sequences complementary thereto :-

5'· -CC ATCCC AATTATAATATCCAACTC-3 ' (SEQ ID NO: 1)

5'· -TGATTATTTCTTGGCCTGTGTGT-3 ' (SEQ ED NO: 2)

5'· -TGGCTCTAGCCGTTCGATTA-3 ' (SEQ ID NO: 3)

5'· -AAGGC AACAGCAAATTCTAGG-3 ' (SEQ ID NO: 4)

5'· -GGAATAATGTTTCGACCACTAAGC-3 ' (SEQ ID NO: 5)

5'- -TGCCTGAGATGGGTATTAGGAT-3 ' (SEQ ID NO: 6)

5'· -ATC ATCAGAACGCCTTATTAGC-3 ' (SEQ ID NO: 7)

5'- - AGGTTCGTCCTTTTGGTGTA-3 ' (SEQ ID NO: 8)

5'- -TGAGACCTCC AACAAATACTAGC-3 ' (SEQ ID NO: 9)

5'- -CTCTATGGC AGAAGGTAGTCAG-3 ' (SEQ ID NO: 10)

The applications of the multiplex PCR kit according to the present invention include the authentication of Halal, kosher, vegetarian and genetically modified foods as well as the analysis of forensic, ecological and archaeological specimens of the species said above.

A pair of primer sequences represented by the sequences (SEQ ID NO: 1) and (SEQ ID NO: 2) or the base sequence complementary thereto are used for the amplification of pig DNA targets which produce 141 bp of amplicon size. In detail, primer sequence represented by sequence (SEQ ID NO: 1) is the forward species-specific mitochondrially encoded NADH dehydrogenase 5 (ND5) primer and primer sequence represented by sequence (SEQ ID NO: 2) is the reverse species-specific mitochondrially encoded NADH dehydrogenase 5 (ND5) primer. A pair of primer sequences represented by the sequences (SEQ ID NO: 3) and (SEQ ID NO: 4) or the base sequence complementary thereto are used for the amplification of dog DNA targets which produce 163 bp of amplicon size. In detail, primer sequence represented by sequence (SEQ ID NO: 3) is the forward species-specific ATP synthase 6 (ATPase 6) primer and primer sequence represented by sequence (SEQ ID NO: 4) is the reverse species-specific ATP synthase 6 (ATPase 6) primer. A pair of primer sequences represented by the sequences (SEQ ID NO: 5) and (SEQ ID NO: 6) or the base sequence complementary thereto are used for the amplification of cat DNA targets which produce 172 bp of amplicon size. In detail, primer sequence represented by sequence (SEQ ED NO: 5) is the forward species-specific Cytochrome b primer and primer sequence represented by sequence (SEQ ED NO: 6) is the reverse species-specific Cytochrome b primer.

A pair of primer sequences represented by the sequences (SEQ ID NO: 7) and (SEQ ED NO: 8) or the base sequence complementary thereto are used for the amplification of rat DNA targets which produce 108 bp of amplicon size. In detail, primer sequence represented by sequence (SEQ ED NO: 7) is the forward species-specific ATP synthase 6 (ATPase 6) primer and primer sequence represented by sequence (SEQ ED NO: 8) is the reverse species-specific ATP synthase 6 (ATPase 6) primer.

A pair of primer sequences represented by the sequences (SEQ ED NO: 9) and (SEQ ED NO: 10) or the base sequence complementary thereto are used for the amplification of monkey DNA targets which produce 129 bp of amplicon size. In detail, primer sequence represented by sequence (SEQ ID NO: 9) is the forward species-specific mitochondrially encoded NADH dehydrogenase 5 (ND5) primer and primer sequence represented by sequence (SEQ ED NO: 10) is the reverse species- specific mitochondrially encoded NADH dehydrogenase 5 (ND5) primer.

The detail of each primer targeted species, genes and amplicon size of each primer (SEQ ID NO: 1 to SEQ ID NO: 10) is summarized in table 2 as below. Table 2

In one specific embodiment, the multiplex PCR kit according to the present invention is more reliable and highly specific in detecting positive samples as the species- specificity was checked against plenty of commercial meat and fish species and no cross-species amplification was detected. Besides, the presence of the target nucleic acids can be detected at concentrations as low as 0.01-0.02 ng/μΐ.

The multiplex PCR kit according to the present invention provides a rapid analysis from nucleic acid extraction to agarose gel electrophoresis which can obtain results in less than 6 hours and can be used with many types of sample matrices including raw food, processed food, feed, tissue and etc.

To further illustrate the details on utilizing the multiplex PCR kit according to the present invention in greater details and not by way of limitation, the following example will be given. EXAMPLE 1

Multiplex PCR in 25μ1 reaction volume containing: 1U GoTaq Flexi DNA Polymerase (Promega, Madison, USA); 5μ1 of 5X GoTaq Flexi Buffer; 200μΜ each of dNTP; 1.5mM MgCl₂; 2.5μ1 of total DNA and 0.2-0.4μΜ primers.

The 2.5μ1 of total DNA are selected from the group consists of the DNA of the pig (Sus scrofa), dog (Canis lupus familiaris), cat (Felis catus), rat (Rattus rattus) and monkey (Macaca fascicularis). While the 0.2-0.4 μΜ primers are selected from the group consists of SEQ ID NO: 1 to SEQ ID NO: 10.

After conducting the Multiplex PCR reaction, the amplified PCR products are analyzed by Bio-Rad Experion Automated Electrophoresis Station using Experion DNA IK Analysis Kit and the results of the amplified PCR products is listed in figure

1 to figure 12.

Figure 1 shows the gel image for the conducted Multiplex PCR reaction, while figure

2 to figure 12 are the corresponding electropherogram plot results of the said Multiplex PCR reaction from lane number 1 to lane number 11. In Figure 1, the gel image: lane L is a DNA Ladder used as size reference; lane 1 is a negative control; lane 2 is a duplex PCR of dog and pig; lane 3 is a triplex PCR of dog, pig and rat; lane 4 is a triplex PCR of cat, pig and rat; lane 5 is a tetraplex PCR of cat, dog, monkey and rat; lane 6 is a multiplex PCR of cat, dog, pig, monkey and rat; lane 7 is a PCR of rat; lane 8 is a PCR of monkey; lane 9 is a PCR of pig; lane 10 is a PCR of dog; and lane 11 is a PCR of cat.

Figure 2 is a corresponding electropherograms of lane 1 illustrates the peaks for negative control with amplicon size of 1500 bp and 15 bp. Figure 3 is a corresponding electropherograms of lane 2 illustrates the peaks for duplex PCR of dog (amplicon size 163 bp) and pig (amplicon size 141 bp). Figure 4 is a corresponding electropherograms of lane 3 illustrates the peaks for triplex PCR of dog (amplicon size 163 bp), pig (amplicon size 141 bp) and rat (amplicon size 108 bp). Figure 5 is a corresponding electropherograms of lane 4 illustrates the peaks for triplex PCR of cat (amplicon size 172 bp), pig (amplicon size 141 bp) and rat (amplicon size 108 bp). Figure 6 is a corresponding electropherograms of lane 5 illustrates the peaks for tetraplex PCR of cat (amplicon size 172 bp), dog (amplicon size 163 bp), monkey (amplicon size 129 bp) and rat (amplicon size 108 bp). Figure 7 is a corresponding electropherograms of lane 6 illustrates the peaks for multiplex PCR of cat (amplicon size 172 bp), dog (amplicon size 163 bp), pig (amplicon size 141 bp), monkey (amplicon size 129 bp) and rat (amplicon size 108 bp). Figure 8 is a corresponding electropherograms of lane 7 illustrates the peak for PCR of rat (amplicon size 108 bp). Figure 9 is a corresponding electropherograms of lane 8 illustrates the peak for PCR of monkey (amplicon size 129 bp). Figure 10 is a corresponding electropherograms of lane 9 illustrates the peak for PCR of pig (amplicon size 141 bp). Figure 11 is a corresponding electropherograms of lane 10 illustrates the peak for PCR of dog (amplicon size 163 bp). Figure 12 is a corresponding electropherograms of lane 11 illustrates the peaks for PCR of cat (amplicon size 172 bp).

To further illustrate the details on the sensitivity of the multiplex PCR kit according to the present invention in greater details and not by way of limitation, the following example will be given. EXAMPLE 2

In order to evaluate the sensitivity, multiplex PCRs by using primers selected from the group consists of SEQ ID NO: 1 to SEQ ID NO: 10 were performed with 10, 5, 1, 0.5, 0.2, 0.1, 0.05, 0.02 and 0.01 ng of DNA template from each species in a common reaction mixture. The DNA band patterns of lane 2-10 in figure 13 shows five bands corresponding to the five species selected from the group consists of the pig, dog, cat, rat and monkey.

Figure 13 shows the gel image for the sensitivity of Multiplex PCR, while figure 14 to figure 24 are the corresponding electropherogram plot results of the said Multiplex PCR from lane number 1 to lane number 11. In Figure 13 the gel image: lane L is a DNA Ladder used as size reference, while the band patterns of lanes 1 to 11 are obtained from 0, 10, 5, 1, 0.5, 0.2, 0.1, 0.05, 0.02, 0.01 and 0 ng of DNA from five meat species (names cat, dog, pig, monkey and rat).

As referring to figure 13 and figure 14, lane 1 is a negative control of 0 ng DNA template of the said five meat species; as referring to figure 13 and figure 15, lane 2 is a multiplex PCR with 10 ng DNA template of the said five meat species; as referring to figure 13 and figure 16, lane 3 is a multiplex PCR with 5 ng DNA template of the said five meat species; as referring to figure 13 and figure 17, lane 4 is a multiplex PCR with 1 ng DNA template of the said five meat species; as referring to figure 13 and figure 18, lane 5 is a multiplex PCR with 0.5 ng DNA template of the said five meat species; as referring to figure 13 and figure 19, lane 6 is a multiplex PCR with 0.2 ng DNA template of the said five meat species; as referring to figure 13 and figure 20, lane 7 is a multiplex PCR with 0.1 ng DNA template of the said five meat species; as referring to figure 13 and figure 21, lane 8 is a multiplex PCR with 0.05 ng DNA template of the said five meat species; as referring to figure 13 and figure 22, lane 9 is a multiplex PCR with 0.02 ng DNA template of the said five meat species; as referring to figure 13 and figure 23, lane 10 is a multiplex PCR with 0.01 ng DNA template of the said five meat species; as referring to figure 13 and figure 24, lane 11 is repeated with a negative control of 0 ng of DNA template of the said five meat species.

In lane 10, the bands for cat and pig were extremely faded but those for dog, monkey and rat were clearly observed. So the limit of detection for cat and pig was 0.02 ng and that for dog, monkey and rat was 0.01 ng.

Conclusion

The five pairs of species-specific primers are developed to target the intra-species conserved and interspecies hyper variable regions of mitochondrial Cytochrome b (cyt b), ATPase 6 and ND5 genes which amplify short-length amplicons in the range of 108 to 172 bp. Since the short-length nucleic acid targets are extraordinarily stable under processing conditions and mitochondrial genes are present in multiple copies, the assay would increase the chances of target detection even in the degraded and extremely processed meats and food products.

The multiplex PCR kit developed according to the present invention conveniently detected five meat species forbidden in Islamic foods with 0.01 to 0.02 ng sensitivity, clearly shows its appeal in halal food industry and halal regulatory bodies. Instead of single species, the detection of five different species in a single assay platform would clearly cut the analysis cost by at least five times. List of references

Sahilah, AM; Norhayati, Y; Norrakiah, AS; Arninah, A; Wan Aida, W. 2011. Halal authentication of raw meats using PCR amplification of mitchondrial DNA. International Food Research Journal, 18(4): 1489-1491. Chandrika, M; Zainon, MN; Maimunah, M; Lesley, MB; Jinap, S; Son R, 2009. Meat species identification and Halal authentication analysis using mitochondrial DNA. Weatherall, D.J.; Clegg, J.B. Meat Science. 57-61.

Matsunaga, T; Chikuni, K; Tanabe, R; Muroya, S; Shibata, K; Yamada, J; Shinmura, Y, 1999. A quick and simple method for the identification of meat species and meat products by PCR assay. Meat Science, 51: 143-148.

Dalmasso, A; Fontanella, E; Piatti, P; Civera, T; Rosati, S; Bottero, MT, 2004. A multiplex PCR assay for the identification of animal species in feedstuffs. Molecular and Cellular Probes. 18(2): 81-87.

Di Pinto, A; Forte, VT; Conversano, MC; Tantillo, GM, 2005. Duplex polymerase chain reaction for detection of pork meat in horse meat fresh sausages from Italian retail sources. Food Control. 16(5): 391-394. Luo, J; Wang, J; Bu, D; Dan, L; Wang, L; Wei, H; & Zhou, L, 2008. Development and Application of a PCR Approach for Detection of Bovis, Sheep, Pig, and Chicken Derived Materials in Feedstuff. Agricultural Sciences in China, 7(10): 1260-1266. Ghowati, S; Nassiri, MR; Mirhoseini, SZ; Moussavi, AH; Javadmanesh, A. 2009. Fraud identification in industrial meat products by multiplex PCR assay. Food Control. 20(8): 696-699.

Yin, RH; Bai, WL; Wang, JM; Wu, CD; Dou, QL; Yin, RL; Luo, GB, 2009. Development of an assay for rapid identification of meat from yak and cattle using polymerase chain reaction technique. Meat Science, 83(1), 38-44.

Zha, D; Xing, X; Yang, F, 2010. A multiplex PCR assay for fraud identification of deer products. Food Control, 21(10): 1402-1407.

Kitpipit, T; Sittichan, K; Thanakiatkrai, P, 2014. Direct-multiplex PCR assay for meat species identification in food products. Food Chemistry, 163: 77-82.

Ali ME, Razzak, MA; Hamid, SB A, 2014. Multiplex PCR in Species Authentication: Probability and Prospects— A Review. Food Analytical Methods, doi: 10.1007/s 12161-014-9844-4.

Murugaiah, C; Noor, ZM; Mastakim, M; Bilung, LM; Selamat, J; Radu, S, 2009. Meat species identification and Halal authentication analysis using mitochondrial DNA. Meat science. 83(1): 57-61.

Claims

WE CLAIM:

1. A multiplex polymerase chain reaction kit for the detection of pig, dog, cat, rat and monkey derived materials for halal authentication consisting of 5 pairs of species-specific primers comprising base sequence represented by sequences (SEQ ID NO: 1) to (SEQ ID NO: 10) or the base sequences complementary thereto characterised in that:

the base sequences (SEQ ID NO:l) and (SEQ ID NO:2) or the base sequence complementary thereto are used as a primer pair for the amplification of pig DNA targets which produce 141 bp of amplicon size; the base sequences (SEQ ED NO:3) and (SEQ ID NO:4) or the base sequence complementary thereto are used as a primer pair for the amplification of dog DNA targets which produce 163 bp of amplicon size; the base sequences (SEQ ID NO: 5) and (SEQ ID NO: 6) or the base sequence complementary thereto are used as a primer pair for the amplification of cat DNA targets which produce 172 bp of amplicon size; the base sequences (SEQ ID NO: 7) and (SEQ ID NO: 8) or the base sequence complementary thereto are used as a primer pair for the amplification of rat DNA targets which produce 108 bp of amplicon size; and the base sequences (SEQ ID NO: 9) and (SEQ ID NO: 10) or the base sequence complementary thereto are used as a primer pair for the amplification of monkey DNA targets which produce 129 bp of amplicon size.

2. The multiplex polymerase chain reaction kit according to claim 1, wherein the base sequence (SEQ ID NO: 1) is the forward species-specific primer and the base sequence (SEQ ID NO: 2) is the reverse species-specific primer for the amplification of pig mitochondrial encoded NADH dehydrogenase 5, ND5 gene.

The multiplex polymerase chain reaction kit according to claim 1, wherein the base sequence (SEQ ED NO: 3) is the forward species-specific primer and the base sequence (SEQ ID NO: 4) is the reverse species-specific primer for the amplification of dog ATP synthase 6, ATPase 6 gene.

The multiplex polymerase chain reaction kit according to claim 1, wherein the base sequence (SEQ ED NO: 5) is the forward species-specific primer and the base sequence (SEQ ED NO: 6) is the reverse species-specific primer for the amplification of cat Cytochrome b gene.

The multiplex polymerase chain reaction kit according to claim 1, wherein the base sequence (SEQ ED NO: 7) is the forward species-specific primer and the base sequence (SEQ ED NO: 8) is the reverse species-specific primer for the amplification of rat ATP synthase 6, ATPase 6 gene.

The multiplex polymerase chain reaction kit according to claim 1, wherein the base sequence (SEQ ID NO: 9) is the forward species-specific primer and the base sequence (SEQ ID NO: 10) is the reverse species-specific primer for the amplification of monkey mitochondrial encoded NADH dehydrogenase 5, ND5 gene.

The multiplex polymerase chain reaction kit according to claim 1, wherein the sensitivity of the multiplex PCR kit for detection of pig, dog, cat, rat and monkey derived materials is as low as 0.01 ng to 0.02 ng.

Use of 5 pairs of primers comprising base sequence represented by sequences (SEQ ED NO: 1) to (SEQ ID NO: 10) or the base sequences complementary thereto for detection of pig, dog, cat, rat and monkey derived materials for halal authentication.