WO2016117999A1

WO2016117999A1 - Dna extraction method on suture samples for porcine trace polymerase chain reaction (pcr) test

Info

Publication number: WO2016117999A1
Application number: PCT/MY2016/000002
Authority: WO
Inventors: Mohd Saifuddeen Bin Sh. Mohd Salleh Sh.; Farhani Binti Zarmani Nur; Anuar Bin Ramli Mohd
Original assignee: University Of Malaya
Priority date: 2015-01-21
Filing date: 2016-01-20
Publication date: 2016-07-28

Abstract

The present invention disclosed a method to trace porcine DNA in a catgut suture by extracting the DNA from the catgut suture and analyzing the extracted DNA. The method of tracing porcine DNA in a catgut suture comprising steps of: homogenizing the catgut suture into a mixture of plant tissue lysis buffer and proteinase K; separating the DNA from the homogenous solution by using the column; and analyzing the extracted DNA by using polymerase chain reaction, PCR.

Description

DNA EXTRACTION METHOD ON SUTURE SAMPLES FOR PORCINE TRACE POLYMERASE CHAIN REACTION (PCR) TEST

FIELD OF THE INVENTION

The present invention relates to a method of tracing porcine DNA in a catgut suture. More specifically, the present invention relates to a method to trace porcine DNA in a catgut suture by extracting the DNA from the catgut suture and analyzing the extracted DNA. BACKGROUND OF THE INVENTION

Of all medical devices applied as implants in the human body, surgical sutures constitute the largest groups of materials with a huge market share exceeding USD 1.3 billion annually (Bloom & Goldberg, 2007). Suture is the most widely used material in wound closure and has been in use for many centuries (Pillai & Sharma, 2010). Both catgut and bonewax are used in wound closure in a surgical procedure.

Catgut suture, whether plain or chromic, is a natural, collagen-based absorbable suture (Hussey & Bagg, 2011), derived from purified connective tissue (Tajirian & Goldberg, 2010) of the twisted small intestines of herbivorous animals (Martin-Bates, 2008). Catgut is still used in modern surgery and accounts for nearly half of all sutures and ligatures. Other forms of sutures are mainly non-absorbable materials such as silk, linen, steel wire and synthetics such as polyester, and nylon (Martin-Bates, 2008). Catgut suture is recognized for its great toughness and tenacity (Pillai & Sharma, 2010). Catgut has the advantage of rapid absorption without the necessity of suture removal (Hussey & Bagg, 201 1). Hence, catgut is practical in pediatric patients, in securing grafts (Moy, Waldman, & Hein, 1992) or in wounds that are difficult to remove sutures (Hochberg, Meyer, & Marion, 2009).

From the first applications in orthopedic (John R. & Virginia neeland, 1950) and cranial surgery (Douglas, 1953) in the 1950s, beeswax has been used routinely to seal sternal edges after median sternotomy in order to reduce oozing (Casha et al., 2001). Because of its texture, puttylike plastic properties and temperature responsiveness, it has almost ideal qualities for application in bony structures to seal oozing from open bone marrow (Prziborowski, Hartrumpf, Stock, Kuehnel, & Albes, 2008). Bonewax is manufactured from sterilized, white-bleached honeybees' wax (cera alba). The composition of 1 g of bonewax are 750 mg cera alba, 150 mg paraffinum sol. and 100 mg isopropylis palmitate (Prziborowski et al., 2008). Under the microscope, body parts of bees such as mandibles, wings, and legs can be found in commercially available bonewax. However, the sterilized product is not considered to carry harmful microorganisms (Nelson, Buxton, Luu, & Rissing, 1990). Issue on halal authenticity is one of the major concerns for Muslims today. In Islam, an important factor for Muslim consumers is whether a product is halal (lawful) or haram (unlawful) (Ramin Jorfi et al., 2012). Demands for products with halal certification are escalating, in line with the growth of population (Ahmad Nizam Abdullah, 2006).

Most of the issues pertaining to halal are related to the presence of porcine-based materials. Another common halal issue is the way in which animals are slaughtered which must conform to Islamic shariah. Islam prohibits pork and all porcine derivatives from being consumed or utilised by Muslims. Therefore, a precise analytical method has to be developed in order to ensure halal authenticity of products so that Muslim consumers would not be exposed to fraud and deception. The authentication of raw materials and finished products as well as the detection of various forms of product adulteration are predominant for both consumers and industries (Ahmad Nizam Abdullah, 2006). Recently, there is a great need for product analysis and authentication due to the increasing health concerns and sensitivity among the consumers (Ramin Jorfi et al., 2012).

Therefore, porcine detection in various products has been an important subject of study in many countries, especially where religious laws prohibit the consumption of pork products (Ramin Jorfi et al., 2012). Presently, numerous physicochemical and genetic techniques have been developed for detection of porcine such as Fourier transform infrared (FTIR) spectroscopy (Che Man et al., 2010), chromatography techniques, electronic nose (EN), polymerase chain reaction (PCR) (A.A. Aida et al., 2005) and restriction-enzyme fragment length polymorphism (RFLP).

In order for an animal-based product to be halal, the animal itself must be halal (religiously acceptable), and must be slaughtered according to the Islamic method (halal slaughter) ( hadijah Nakyinsige et al., 2012). Additionally, contamination with haram substances should be avoided throughout the manufacturing process and the product must not contain any haram ingredient. Methods that have been for halal detection include chromatography (J.M. N. Marikkar, et al., 2005), DNA-based technology (Aida A. A., Y. B. Che Man, A. R. Raha & R. Son, 2007), spectroscopic method (Y.B. Che Man, Syahariza, Mirghani, Jinap, & J. Bakar, 2005) followed by a rapid, short column chromatography technique, electronic nose (Nurjuliana, Che Man, & Mat Hashim, 2010).

There exists an extensive literature on the verification of halal food product, cosmetics and pharmaceuticals. The determination of halal status in food products is often debated (Mohammad Aizat Jamaludin et al., 2011) as a result of advancements in the area of science and technology in terms of source of material, preparation, processing, transmission of food from the farm until it is served on the table. However, there is still no specific study on halal medical devices.

In the medical device industry, catgut sutures are often used as a tool for wound closure and ligation on a wide area. As it is made of animal source, this situation has resulted in increased concerns regarding the compliance of such products from the halal perspective. With regards to this issue, the development of various instrumental techniques is in great demand for halal authentication for identifying the origins of species which are used as catgut sutures and to ascertain if it originates from porcine, bovine or plants. In order to determine the DNA content of the catgut sutures, a specific method to extract DNA from catgut sutures must be developed. Currently, there is still no specific method to extract DNA from catgut sutures. Thus, it would be advantageous to develop a method that can extract the DNA from catgut sutures to identify the origins of species which are used as catgut sutures. The present invention is addressed to the abovementioned issues.

SUMMARY

An object for the present invention is to provide a method for tracing porcine DNA in a catgut suture. Surgical sutures represent the largest groups among all medical devices applied as implants in the human body. Catgut suture is the most widely used material in wound closure and has been in use for many centuries in a surgical procedure. The absorption profile of catgut suture make it practical in pediatric patients, in securing grafts or in wounds from which it is difficult to remove the sutures. In the catgut suture production chain today, collagen is usually extracted from submucosa layer of small intestine of sheep or cattle. Clearly collagen can be derived from both permissible (halal) and non-permissible (haram) sources. Hence, developing a method for tracing porcine DNA in a catgut suture is important to Halal authentication industry.

The method for tracing porcine DNA in a catgut suture according to present invention comprising steps of: cutting a catgut suture into small pieces; freezing the cut catgut suture; grinding the frozen catgut suture; adding the grounded catgut suture into lysis buffer to become a mixture; rigorously mixing the mixture to homogenize the mixture; adding proteinase into the homogenized mixture and incubating the homogenized mixture at 65 °C for 5 hours with frequent mixing during incubation to assure complete digestion of the catgut suture in the homogenized mixture; centrifuging the homogenized mixture to precipitate any insoluble or undigested substances; transferring out the supernatant which consisting the DNA; adding the genomic binding buffer into the supernatant and mixing until becoming a homogenous solution; incubating the homogenous solution at 65 °C for 10 minutes; mixing an absolute ethanol into the homogenous solution; separating the DNA from the homogenous solution by using the column; eluting the DNA from the membrane of the column to get the extracted DNA; analyzing the extracted DNA by using polymerase chain reaction, PCR. These and other objects, features and advantages of the present invention will be readily apparent from the following description.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

The accompanying drawings/figures are included to provide a further understanding of the present invention and 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 the operational steps of a method for tracing porcine DNA in a catgut suture according to the present invention;

Figure 2 shows PCR result of EB1 sample extracted with the first extraction method. 1) Pork trace positive control, 2) Plant/amplification control, 3) Vertebrate positive control, 4) Mix pork and plant positive control, 5 - 7) Negative control, M) Porcine marker, 8) EB1 with EAC sample, 9) EB1 with EAC sample at 1 :10 dilution, and 10) EB1 with EAC sample at 1 :100;

Figure 3 shows PCR result of EB2 sample extracted with the method according to the present invention. 1) Pork trace positive control, 2) Plant/amplification control, 3) Vertebrate positive control, 4) Mix pork and plant positive control, 5 - 7) Negative control, M) Porcine marker, 8) EB2 with EAC sample, 9) EB2 with EAC sample at 1 :10 dilution, and 10) EB2 with EAC sample at 1 :100 dilution; Figure 4 shows PCR result of EB3 sample extracted with phenol chloroform extraction method. 1) Pork trace positive control, 2) Plant/amplification control, 3) Vertebrate positive control, 4) Mix pork and plant positive control, 5 - 7) Negative control, M) Porcine marker, 8) EB3 sample with EAC, 9) EB3 sample with EAC at 1 :10 dilution and 10) EB3 sample with EAC at 1 :100 dilution;

Figure 5 shows PCR result of CGI sample extracted with the first extraction method. 1) Pork trace positive control, 2) Plant/amplification control, 3) Mix pork and plant positive control, 4) Vertebrate positive control, 5 - 7) Negative control, M) Porcine marker, 8) CGI sample, 9) CGI sample at 1 :10 dilution and 10) CGI sample at 1 :100 dilution; Figure 6 shows PCR result of CG2 sample extracted with the method according to the present invention. 1) Pork trace positive control, 2) Plant/amplification control, 3) Vertebrate positive control, 4) Mix pork and plant positive control, 5 - 7) Negative control, M) Porcine marker, 8) CG2 sample, 9) CG2 sample at 1 :10 dilution and 10) CG2 sample at 1 :100 dilution; and

Figure 7 shows PCR result of CG3 sample extracted with phenol chloroform extraction method. 1) Pork trace positive control, 2) Plant/amplification control, 3) Vertebrate positive control, 4) Mix pork and plant positive control, 5 - 7) Negative control, M) Porcine marker, 8) CG3 sample, 9) CG3 sample at 1 :10 dilution and 10) CG3 sample at 1 : 100 dilution.

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 skilled in the art to practice the present invention 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 represents a method of tracing porcine DNA in a catgut suture. As discussed before, catgut suture is a natural collagen-based absorbable suture derived from purified connective tissue of the animal, thus, the main object of the present invention is to invent a novel and most effective way to extract the animal originated DNA from the catgut suture and once the DNA is extracted from the catgut suture, the extracted DNA can be analyzed by porcine trace polymerase chain reaction, PCR analysis. Figure 1 is an example of a representative flowchart of a method in the present invention. This flowchart is merely an illustration and the scope of the claims herein should not be limited. One of ordinary skilled in the art will recognize other variations, modifications and alternatives. Referring to figure 1, the method of tracing porcine DNA in a catgut suture according to the present invention, indicated by reference numeral (10) comprising steps of: cutting a catgut suture into small pieces, indicated by reference numeral (11); freezing the cut catgut suture, indicated by reference numeral (12); grinding the frozen catgut suture, indicated by reference numeral (13); adding the grounded catgut suture into lysis buffer to become a mixture, indicated by reference numeral (14); rigorously mixing the mixture to homogenize the mixture, indicated by reference numeral (15); adding proteinase K into the homogenized mixture and incubating the homogenized mixture at 65 °C for 5 hours with frequent mixing during incubation to assure complete digestion of the catgut suture in the homogenized mixture, indicated by reference numeral (16); centrifuging the homogenized mixture to precipitate any insoluble or undigested substances, indicated by reference numeral (17); transferring out the supernatant which consisting the DNA, indicated by reference numeral (18); adding the genomic binding buffer into the supernatant and mixing until becoming a homogenous solution, indicated by reference numeral (19); incubating the homogenous solution at 65 °C for 10 minutes, indicated by reference numeral (20); mixing an absolute ethanol into the homogenous solution, indicated by reference numeral (21); separating the DNA from the homogenous solution by using the column, indicated by reference numeral (22); eluting the DNA from the membrane of the column to get the extracted DNA, indicated by reference numeral (23); analyzing the extracted DNA by using polymerase chain reaction, PCR, indicated by reference numeral (24).

In any analytical process, sample preparation refers to the ways in which a sample is treated prior to its analysis. Sample preparation is a very important step in most analytical techniques including but not limited to the DNA analysis because the techniques are often not responsive to the analyte in its in-situ form or the results are distorted by interfering species. Catgut suture is a long and thin thread, thus, catgut suture must undergo a suitable sample preparation process before analysis. As indicated in figure 1 , catgut suture is cut into small pieces (11), then, the cut catgut suture is frozen (12) and ground (13) into a fine powder. These steps are the initial steps to prepare the catgut suture sample. Grinding catgut suture sample into fine powder can help to homogenize the catgut suture sample so that it is easier for the sample to be added into a lysis buffer.

Still referring to figure 1, a lysis buffer is added into the catgut suture samples to become a mixture (14) and the mixture is rigorously mixed by vortexing the mixture for 30 seconds to homogenize the mixture (15). In a specific embodiment, the lysis buffer used according to the present invention is selected from the plant tissue lysis buffer. Then, proteinase K is added into the homogenized mixture and the homogenized mixture is incubated at 65 °C for 5 hours with frequent mixing during incubation to assure complete digestion of the catgut suture in the homogenized mixture (16).

After incubation, the homogenized mixture is centrifuged at 14,000 x g for 5 min to precipitate any insoluble or undigested substances (17). Then, a supernatant consists of the DNA is transferred into a clean microcentrifuge tube (18). A genomic binding buffer is added into the supernatant and rigorously mixed until a homogenous solution is obtained (19). In a specific embodiment, the genomic binding buffer used according to the present invention is selected from the plant genomic binding buffer. Then, the homogenous solution is incubated at 65°C for 10 minutes (20). An absolute ethanol is immediately and thoroughly added into the homogenous solution (21). The DNA is separated out from the homogenous solution by using the column (22). In operation step (22), the homogenous solution is transferred into a column assembled in a clean collection tube. The tube is then centrifuged at 10,000 x g for 1 minute. Flow through is discarded. The procedure is repeated until all supernatant are passed through the column. The column is washed with wash buffer and is centrifuged at 10,000 x g for 1 minute. Flow throw is discarded. Washing is repeated when color stains from sample remained on the column membrane. The column is centrifuged at 10,000 x g for 1 minute to remove residual ethanol. All traces of ethanol are completely removed because any residual ethanol can affect the quality of DNA and may eventually inhibit reactions of enzymes. The column is placed into a clean microcentrifuge tube. Preheated elution buffer is added directly onto the column membrane and stand for 2 minutes. The column is centrifuged at 10,000 x g for 1 minute to elute DNA (23). Finally, the extracted DNA is analyzed by using polymerase chain reaction, PCR to detect the porcine traces (24).

To further illustrate the present invention in greater details and not by way of limitation, the following examples will be given. In the following examples, 3 different extraction methods are illustrated for extracting the DNA in catgut suture and bonewax. The examples are to prove that the method disclosed according to the present invention is the most effective method for extraction of DNA from catgut sutures.

EXAMPLE 1

First extraction method

For catgut suture sample, labeled as CGI, 50mg of suture were cut into small pieces with a sterile scalpel. It was then frozen and ground with a mortar and pestle and then transferred into a clean microcentrifuge tube. For the bonewax sample, labeled as EBl, 200mg of sample were excised out and 300μ1 of 0.5% Tween 20 were added to the sample in a microcentrifuge tube. The sample was heated to 90°C for 10 minutes to melt the wax. The sample was agitated and maintained at 55°C.

700μ1 Buffer FL and 20μ1 Proteinase were added into the CGI while 400μ1 Buffer FL and 20μ1 Proteinase K were added into the EBl sample. They were rigorously mixed by vortexing in order to acquire a homogenous solution. The solution was then incubated at 65°C for 5 hours instead of 30 minutes as suggested in the product manual with frequent mixing during incubation to assure complete lysis of the catgut suture sample. Then, the solution was centrifuged at 12,000 x g for 5 minutes to pellet the cell debris and contaminants. 450μ1 of cleared supernatant was then transferred to a clean microcentrifuge tube. After that, 680μ1 (which is 1.5 volumes) of food genomic binding buffer (Buffer FB) was added and rigorously mixed by pulsed- vortexing until a homogenous solution was attained. The solution was then incubated at 65°C for 10 minutes.

350μ1 of absolute ethanol was added to the mixture. It was then immediately and rigorously mixed by pulsed-vortexing in order to acquire a homogenous solution to deter any uneven nucleic acid precipitation due to high concentration of local ethanol. Then, it was centrifuged at 5,000 x g for 1 minute. Approximately 650μ1 of supernatant was transferred into a column assembled in a clean collection tube before it was centrifuged at 5,000 x g for 1 minute. Then, flow through was discarded. The procedure was repeated until all supernatant were passed through the column.

The column was washed with 500μ1 Wash Buffer 1 and 2 with 5,000 x g for 1 minute centrifugation between the steps and flow through was discarded. The column was washed again with 500μ1 Wash Buffer 2 and centrifuged at greatest speed for 3 minutes in order to completely remove the ethanol traces. The column was placed into a clean microcentrifuge tube. 30μ1 of preheated Elution Buffer was added directly onto the column membrane and stood for 2 minutes. In order to get complete elution, the elution buffer was ensured to dispense right onto the center of the membrane. The column was centrifuged at 5,000 x g for 1 minute to elute the DNA of catgut suture. The DNA was stored at -20°C to prevent DNA from degrading in the absence of buffering agent.

All DNA samples extracted were quantified using Nanodrop vlOOO Machine. EXAMPLE 2

Second extraction method - A method according to present invention

The second extraction method is based on the method disclosed according to the present invention Similarly, 50 mg of catgut suture (CG 2) sample was cut into small pieces with a clean scalpel. It was then frozen and ground with a mortar and pestle. For the bonewax sample (EB2), 200mg was excised out and transferred into a clean microcentrifuge tube.

280μ1 of plant tissue lysis buffer was added to the samples and they were rigorously mixed by vortexing the tube for 30 seconds to have a homogenous solution. 20μ1 of Proteinase K was added and mix rigorously by inverting tube. It was then incubated at 65°C for 5 hours with frequent mixing during incubation to assure complete digestion of sample. After incubation, it was then centrifuged at 14,000 x g for 5 min to precipitate any insoluble or undigested substances. 300μ1 of supernatant consisting the DNA was transferred into a clean microcentrifuge tube.

600μ1 of plant genomic binding buffer was added and rigorously mixed until a homogenous solution was obtained. Then, it was incubated at 65 °C for 10 minutes. 200μ1 of absolute ethanol was added into the tube and mixed immediately and thoroughly.

650μ1 of mixture was then transferred into a column assembled in a clean collection tube. The tube then was centrifuged at 10,000 x g for 1 minute. Flow through was discarded. The procedure was repeated until all supernatant passed through the column. The column was washed with 650μ1 Wash Buffer and was centrifuged at 10,000 x g for 1 minute. Flow throw was discarded. Washing was repeated when color stains from sample remained on the column membrane. The column was centrifuged at 10,000 x g for 1 minute to remove residual ethanol. All traces of ethanol were completely removed because any residual ethanol can affect the quality of DNA and may eventually inhibit reactions of enzymes.

The column was placed into a clean microcentrifuge tube. 30μ1 of preheated Elution Buffer was added directly onto the column membrane and stood for 2 minutes. The column was centrifuged at 10,000 x g for 1 minute to elute DNA. DNA was stored at -20°C.

All DNA samples extracted were quantified using Nanodrop vlOOO Machine. EXAMPLE 3

Third extraction method - a method of extraction using conventional phenol

This chloroform method is a conventional phenol method that was adopted from Tabanifar et al., (2008). 50 mg of catgut suture (CG 3) and 200 mg of bonewax (EB 3) were used. First, 400μ1 of lysis buffer (lOmM Tris pH8.0, lOmM NaCl, 2% SDS, 39mM DTT) and 0.4mg/ml Proteinase were added. Then, the solution was incubated overnight at 65°C. Equal volume of phenol : chloroform : isoamyl alcohol (25:24:1) solution were added into the sample and the solution was vigorously vortexed.

The solution was then centrifuged at 12,000 x g for 10 minutes. The top aqueous layer was transferred into a new 1.5 ml tube, then 1 volume of chloroform was added. After that, the tube was vortexed and later was centrifuged at 12,000 x g for 10 minutes. The top aqueous layer was transferred into a new 1.5 ml tube before 1/10 volume of 3M sodium acetate was added.

The solution was vortexed and 1 volume of isopropanol was added. The solution was incubated overnight at -20°C. Next, the tube was centrifuged at 14,000 x g for 10 minutes at 4°C. The supernatant was discarded and pellet was washed with ice cold 75% ethanol. Then, the pellet was centrifuged at 14,000 x g for 10 minutes at 4°C. To dry the pellet, it was left in fume hood with cap open. Finally, the pellet was dissolved in 30μ1 of lx Tris EDTA buffer.

All DNA samples extracted were quantified using Nanodrop vlOOO Machine.

EXAMPLE 4

Detection of porcine traces using PCR

Porcine Traces PCR v.2 kit (Profound Kestrel Laboratories) was used to detect porcine traces in the DNA extracted samples with the aid of TProfessional Trio Block (Biometra GmbH) machine. In this protocol, the reaction mix was prepared to make up a volume of 20μ1 per PCR tube reaction as followed (Table 1): Table 1 : Components of the reaction mix and the setup

Ιμΐ of 1 :50 diluted internal amplification control (EAC) was added to check for false negatives in bonewax sample only. As the catgut sample is of vertebrate (animal) origin, the PCR will be able to amplify the vertebrate (animal) band as internal control for catgut samples. Pre-extracted DNA sample were serially diluted at 1 :10 and 1 :100 dilutions and used for the PCR testing. 5μ1 of samples (diluted and non-diluted) was pipetted into the PCR mastermix tube each. The DNA sample was topped up to 25μ1 of total volume with nuclease free water. For negative control, 1 tube was set aside as no template control by adding only water as template. For positive control, 1 tube was set aside for positive control by adding Ιμΐ of Pork DNA as a template.

The PCR tubes were spun down and PCR was run using the following thermal cycling condition listed in table 2: Table 2: Thermal cycler parameters

A 3% of 0.5x TBE agarose gel was prepared. 5μ1 of PCR product was loaded into each well. Ιμΐ of Porcine Trace DNA marker and a lOObp DNA ladder was loaded as well. A full length gel was run (the lower dye was ensured that it reached 90% of the gel length). After the run, the gel was stained with Ethidium Bromide (EtBr) appropriately, the gel was visualized with an UV transilluminator.

Results and Discussions

DNA quantification

Nanodrop vlOOO Machine was used for DNA quantification, with upper limit approximately

(ds DNA). Table 3 shows the DNA extraction result summary and table 4 shows the PCR result analysis summary. The result of table 4 is derived from figure 2 to figure 7.

Table 3: DNA extraction result summary

Table 4: PCR result analysis summary

N/A** - not applicable because animal derivative amplification serves as internal control

The sample of Bonewax is negative for plant, animal (vertebrate) and porcine derivatives. Therefore, amplification control is used to indicate the presence of inhibitors in the sample. Extraction methods using the first extraction method as disclosed in example 1 (EB1) and the method disclosed according to the present invention (10) (EB2) are free from PCR inhibitors as the amplification control produced bands in all three sample dilutions. The phenol chloroform extracted sample (EB3) is PCR inhibited at original concentration, while in its dilutions, the amplification control band re-appeared. Based on the Nanodrop 1000 concentration reading, the Bonewax sample was most suitably extracted using the first extraction method as disclosed in example 1.

The Catgut sample is positive for animals (vertebrate) only and negative for plant and porcine derivatives. The amplification control was not used. Extraction methods using the first extraction method as disclosed in example 1 (CGI) and the method disclosed according to the present invention (10) (CG2) are free from PCR inhibitors as the vertebrate band was produced in all three sample dilutions. The phenol chloroform extracted sample (CG3) is PCR inhibited at all dilutions. Based on the Nanodrop 1000 concentration reading and PCR results, the chromic catgut sample was most suitably extracted using the method disclosed according to the present invention (10).

Conclusion

Halal authentication methods have been used to verify the DNA in food, cosmetics and pharmaceutical products. However, there is still yet specific study conducted on the DNA content of the surgical sutures, which lies in the category of medical devices. Natural surgical sutures, namely, catgut and chromic catgut are still being used and produced in countries like Malaysia, China and India. Even though sutures are directly used onto human body, the halal status of the suture was never being questioned during the surgery or any medical procedure. This issue may not arise due the condition of the patient during treatment (darurah).

Authentication is the process to verify a product as complying with its label description (Dennis, 1998). Authenticity testing and analytical techniques especially for meat products have been developed, each appropriate and specific to deal with a particular problem.(Khadijah Nakyinsige et al., 2012). For the purpose of halal verification of surgical sutures, the three recommended techniques namely, the first extraction method as disclosed in example 1, the method disclosed according to the present invention (10) and conventional phenol: chloroform methods were modified and adopted. These three protocols were crucial to determine the possible and efficient way to extract the DNA from sutures prior to Porcine Trace PCR Analysis. The sample of Bonewax is negative for plant, animal (vertebrate) and porcine derivatives. Therefore, amplification control is used to indicate the presence of inhibitors in the sample. Extraction methods using the first extraction method as disclosed in example 1 (EBl) and the method disclosed according to the present invention (10) (EB2) are free from PCR inhibitors as the amplification control produced bands in all three sample dilutions. The phenol chloroform extracted sample (EB3) is PCR inhibited at original concentration, while in its dilutions, the amplification control band re-appeared. Based on the Nanodrop 1000 concentration reading, the Bonewax sample was most suitably extracted using the first extraction method as disclosed in example 1.

As discussed before, for extraction methods of bonewax suture, first extraction method as disclosed in example 1 (EB1) and the method disclosed according to the present invention (10) (EB2) are free from PCR inhibitors as the amplification control produced bands in all three sample dilutions. The phenol chloroform extracted sample (EB3) is PCR inhibited at original concentration, while in its dilutions, the amplification control band re-appeared. Based on the Nanodrop 1000 concentration reading, the Bonewax sample was most suitably extracted using the method disclosed according to the present invention (10). However, extraction methods of catgut suture using first extraction method as disclosed in example 1 (CGI) and the method disclosed according to the present invention (10) (CG2) are free from PCR inhibitors as the vertebrate band was produced in all three sample dilutions. The phenol chloroform extracted sample (CG3) is PCR inhibited at all dilutions. Based on the Nanodrop 1000 concentration reading and PCR results, the chromic catgut sample was most suitably extracted using the method disclosed according to the present invention (10).

DNA isolation offers the advantage of differentiating among different animal species solely using DNA analysis (Ballin, 2010). In addition, DNA is a stable molecule that permits analysis of processed and heat treated products (Aida et al., 2005), it is present in majority of cells and the information content of DNA is not only greater than that of protein but it can also be extracted from all kinds of tissues (Lockley & Bardsley, 2000). PCR is capable of amplifying very few copies of DNA and its detection limit is much lower than what is observed with protein based assays (Khadijah Nakyinsige et al., 2012). PCR amplification is based on hybridization of specific oligonucleotides to a target DNA and synthesis of million copies flanked by these primers. The simplest PCR strategy applied to evaluate presence of either plant, vertebrate or porcine trace in the surgical suture product is the amplification of DNA fragments, followed by agarose gel electrophoresis for fragment size verification. To successfully detect a species with PCR, adequate genetic markers are chosen to develop the assay (Khadijah Nakyinsige et al., 2012).

There are several steps to consider in order to increase the yield of DNA extracted. Firstly, DNA yield can be increased through sufficient homogenization of the sample. This step is crucial to ensure that the tissues are completely homogenized in lysis buffer. Insufficient sample lysis may also result in low DNA yield. Therefore, it is suggested to mix the sample thoroughly with lysis buffer and Proteinase . Time of incubation may be extended up to 2 hours at 65°C and the amount starting material may be reduced. Sample must be mixed frequently during incubation in absence of a waterbath shaker. Reduction in Proteinase K activity may also decrease DNA yield. In order to optimize Proteinase K activity, make sure to store it at -20°C. Next, the samples also should be stored properly. For this case, the suture samples are stored at room temperature under sterile condition. The column that is not placed at fixed orientation during centrifugation may also lower the DNA yield. In order to troubleshoot this, the column which has a triangle mark on the edge have to be placed at a fixed position during centrifugation at all times. The column also must be ensured to be dried prior to addition of Elution Buffer by spinning it dried at maximum speed for 3 minutes after addition of Wash Buffer 2. Neglecting the step of additional of alcohol may also result in low DNA yield. Therefore, it is suggested that purification with a new sample to be repeated. The factor of low elution efficiency also results in low DNA yield. Therefore, Elution Buffer needs to be pre-heated to 65-70°C before eluting the DNA. After addition of Elution Buffer, it is suggested to incubate the sample at room temperature for 2 minutes. Make sure that Elution Buffer used is a low salt buffer or water with pH range of 7.0 - 8.5. Through conventional method, chloroform extraction is a critical step to increase the recovery of DNA yield (Xin & Chen, 2012). Other than that, purity of DNA is low possibly because of incomplete protein denaturation. Hence, time of incubation has to be extended until lysate clears to increase the purity. DNA purity may also be affected by contamination of RNA. To troubleshoot this, RNase A can be added to the sample as indicated in the protocol. RNase A used must be ensured that it has not been repeatedly frozen and thawed. A fresh stock can also be prepared if it is necessary. Low purity may also be affected by decreasing of Proteinase activity and incomplete sample lysis.

Column clogged happen because of overloading of column or starting material too high. Therefore, sample used must not be more than 30 mg. If any undigested material remains, it can be spun to remove tissue lysate and supernatant is transferred into a new microcentrifuge tube. Another possibility that result in column clogged is that the sample may not homogenize thoroughly. So, it is suggested that the sample to be vortex in lysis buffer prior to the addition of Proteinase K.

DNA smearing may also occur as it sheared during purification. Hence, to control this problem, after addition of lysis buffer and Proteinase , vigorous mixing and pipetting must be avoided. Instead, gently mix them by inverting tube. This problem may also possible because of nuclease contamination. To avoid this, use sterilized glassware, plasticware and wear gloves. Besides that, the tissue must be ensured that it is completely homogenized in lysis buffer and Proteinase K.

Poor performance of eluted DNA in downstream applications may be affected by eluted DNA contains traces of ethanol. Therefore, the column drying step must be carried out prior to elution.

There are a large number of methods for recovery of DNA from formalin-fixed paraffin-embedded specimens. Some authors believe the main obstacle in preparing DNA suitable for PCR amplification is removal from paraffin wax (Coombs, Gough, & Primrose, 1999). A variety of DNA preparation methods and commercial kits are available. However, they are either low throughput, low yield or costly. Extraction of large quantity and high quality DNA is often a limiting factor in genetic analysis of plant traits important to agriculture. Many high throughput methods to isolate DNA are available; however, these methods produce either insufficient amounts or inconsistent quality of DNA (Flagel et al., 2005).

In DNA quantification, DNA concentrations for all samples quality, except Bonewax 3 and Catgut 3 were within acceptable range for PCR by using NanoDrop vlOOO Spectrophotometer. Both samples were negative for porcine traces, but were PCR able.

References

A.A. Aida, Man, Y. B. C, & C.M.V.L. Wong. (2005). Analysis of raw meats and fats of pigs using polymerase chain reaction for Halal authentication. Meat Science, 69(1), 47-52. doi: 10.1016/j .meatsci.2004.06.020

Ahmad Nizam Abdullah. (2006). Perception and Awarness among Food Manufacturers and Marketers on Halal Food in the Klang Valley. Universiti Putra Malaysia.

Aida, A. A., Man, Y. B. C, Wong, C. M. V. L., Raha, A. R., & Son, R. (2005). Analysis of raw meats and fats of pigs using polymerase chain reaction for Halal authentication. Meat Science, 69, 47-52. doi:10.1016/j.meatsci.2004.06.020 Ballin, N. Z. (2010). Authentication of meat and meat products. Meat Science, doi: 10.1016/j .meatsci.2010.06.001

Bloom, B. S., & Goldberg, D. J. (2007). Suture material in cosmetic cutaneous surgery. Journal of Cosmetic and Laser Therapy : Official Publication of the European Society for Laser Dermatology, 9, 41^15. doi:10.1080/14764170601140062 Casha, A. R., Gauci, M., Yang, L., Saleh, M, Kay, P. H., & Cooper, G. J. (2001). Fatigue testing median sternotomy closures. European Journal of Cardio-Thoracic Surgery, 19, 249-253. doi:10.1016/S1010-7940(01)00584-X Che Man, Y. B., Z. Abidin, S., & Rohman, A. (2010). Discriminant Analysis of Selected Edible Fats and Oils and Those in Biscuit Formulation Using FTIR Spectroscopy. Food Analytical Methods, 4(3), 404-409. doi:10.1007/sl2161-010- 9184-y Coombs, N. J., Gough, A. C, & Primrose, J. N. (1999). Optimisation of DNA and RNA extraction from archival formalin-fixed tissue. Nucleic Acids Research, 27, el 2. doi:10.1093/nar/27.16.el2-i

Douglas, B. L. (1953). Clinical observation on the use of absorbable hemostatic bone wax in dental and oral surgery. Oral Surgery, Oral Medicine, Oral Pathology, 6(10), 1 195.

Flagel, L., Christensen, J. R., Gustus, C. D., Smith, K. P., Olhoft, P. M., Somers, D. A., & Matthews, P. D. (2005). Inexpensive, High Throughput Microplate Format for Plant Nucleic Acid Extraction. Crop Science, doi: 10.2135/cropsci2004.0657

Hochberg, J., Meyer, K. M., & Marion, M. D. (2009). Suture choice and other methods of skin closure. The Surgical Clinics of North America, 89, 627-641. doi:10.1016/j.suc.2009.03.001 Hussey, M., & Bagg, M. (2011). Principles of wound closure. Operative Techniques in Sports Medicine, 19, 206-21 1. doi:10.1053/j.otsm.2011.10.004

J.M. N. Marikkar, Ghazali, H. M., Man, Y. B. C, Peiris, T. S. G., & O.M. Lai. (2005). Distinguishing lard from other animal fats in admixtures of some vegetable oils using liquid chromatographic data coupled with multivariate data analysis. Food Chemistry, 91, 5-14. doi:10.1016/j.foodchem.2004.01.080 John R., G., & Virginia Kneeland, F. (1950). New absorbable hemostatic bone wax: Experimental and clinical studies. Annals of Surgery, 132(6), 1128-1 137.

Khadijah Nakyinsige, Yaakob Bin Che Man, & Awis Qurni Sazili. (2012). Halal authenticity issues in meat and meat products. Meat Science, 91 , 207-214. doi:10.1016/j.meatsci.2012.02.015

Lockley, A. K., & Bardsley, R. G. (2000). DNA-based methods for food authentication. Trends in Food Science and Technology. doi:10.1016/S0924- 2244(00)00049-2

Martin-Bates, A. (2008). Tying all together. Trauma, 10(103), 103-108. doi: 10.1177/1460408608088635 Mohammad Aizat Jamaludin, Mahmood Zuhdi Hj Ab. Majid, & Mohd Anuar Ramli, Nor Nadiha Mohd Zaki, S. A. R. & D. M. H. (201 1). Realisasi Pandangan Fuqaha Tentang Makanan Berasaskan Nilai Saintifik Semasa. In Seminar Hukum Islam Semasa VII (pp. 1-15). Moy, R. L., Waldman, B., & Hein, D. W. (1992). A review of sutures and suturing techniques. The Journal of Dermatologic Surgery and Oncology, 18, 785-795.

Nelson, D. R., Buxton, T. B., Luu, Q. N., & Rissing, J. P. (1990). The promotional effect of bone wax on experimental Staphylococcus aureus osteomyelitis. The Journal of Thoracic and Cardiovascular Surgery, 99, 977-980.

Nurjuliana, M., Che Man, Y. B., & Mat Hashim, D. (2010). Analysis of Lard's Aroma by an Electronic Nose for Rapid Halal Authentication. Journal of the American Oil Chemists' Society, 88(1), 75-82. doi:10.1007/sl 1746-010-1655-1 Pillai, C. K. S., & Sharma, C. P. (2010). Review paper: absorbable polymeric surgical sutures: chemistry, production, properties, biodegradability, and performance. Journal of Biomaterials Applications, 25(4), 291-366. doi: 10.1 177/0885328210384890 Prziborowski, J., Hartrumpf, M., Stock, U. A., Kuehnel, R. U., & Albes, J. M. (2008). Is Bonewax Safe and Does It Help? Annals of Thoracic Surgery, 85, 1002-1006. doi: 10.1016/j .athoracsur.2007.10.036

Ramin Jorfi, Shuhaimi Mustafa, Yaakob B Che Man, Dzulkifly B Mat Hashim, Awis Q Sazili, Abdoreza Soleimani Farjam, L. N. and P. K. (2012). Differentiation of pork from beef, chicken, mutton and chevon according to their primary amino acids content for halal authentication. African Journal of Biotechnology, 11(32), 8160-8166. doi: 10.5897/AJB 11.3777 Tabanifar, B., Salehi, R., Asgarani, E., Faghihi, M., Heidarpur, M., Tajal Sadat, A., & 1. (2008). An Efficient Method for DNA Extraction from Paraffin Wax Embedded Tissues for PCR Amplification of Human and Viral DNA. Iranian Journal of Pathology, 3(4), 173-178. Tajirian, A. L., & Goldberg, D. J. (2010). A review of sutures and other skin closure materials. Journal of Cosmetic and Laser Therapy : Official Publication of the European Society for Laser Dermatology, 12(6), 296-302. doi:10.3109/14764172.2010.538413 Xin, Z., & Chen, J. (2012). A high throughput DNA extraction method with high yield and quality. Plant Methods, doi: 10.1186/1746-481 1-8-26

Y.B. Che Man, Syahariza, Z. A., Mirghani, M. E. S., Jinap, S., & J. Bakar. (2005). Food Chemistry Analysis of potential lard adulteration in chocolate and chocolate products using Fourier transform infrared spectroscopy. Food Chemistry, 90, 815-819. doi: 10.1016/j .foodchem.2004.05.029

Claims

WE CLAIM:

1. A method of tracing porcine DNA in a catgut suture comprising steps of:

i) cutting a catgut suture into small pieces (11);

ii) freezing the cut catgut suture (12);

iii) grinding the frozen catgut suture (13);

iv) adding the grounded catgut suture into lysis buffer to become a mixture (14);

v) rigorously mixing the mixture to homogenize the mixture (15);

vi) adding proteinase into the homogenized mixture and incubating the homogenized mixture at 65 °C for 5 hours with frequent mixing during incubation to assure complete digestion of the catgut suture in the homogenized mixture (16);

vii) centrifuging the homogenized mixture to precipitate any insoluble or undigested substances (17);

viii) transferring out the supernatant consisting the DNA (18);

ix) adding the genomic binding buffer into the supernatant and mixing until becoming a homogenous solution (19);

x) incubating the homogenous solution at 65 °C for 10 minutes (20);

xi) mixing an absolute ethanol into the homogenous solution (21);

xii) separating the DNA from the homogenous solution by using the column (22);

xiii) eluting the DNA from the membrane of the column to get the extracted DNA (23);

xiv) analyzing the extracted DNA by using polymerase chain reaction, PCR (24) characterised in that the lysis buffer used in the step of (14) is selected from the plant tissue lysis buffer and the genomic binding buffer used in the step of (19) is selected from the plant genomic binding buffer.