Simultaneous detection of bovine and porcine DNA in pharmaceutical gelatin capsules by duplex PCR assay for Halal authentication

Abstract

BackgroundIn the pharmaceutical industry, hard- and soft-shelled capsules are typically made from gelatin, commonly derived from bovine and porcine sources. To ensure that pharmaceutical products comply with halal regulations in Muslim countries (no porcine products allowed), development of a valid, reliable, quick, and most importantly, cost-effective tests are of utmost importance.MethodsWe developed a species-specific duplex polymerase chain reaction (PCR) assay targeting 149 bp porcine and 271 bp bovine mitochondrial DNA (mtDNA) to simultaneously detect both porcine and bovine DNA (in one reaction at the same time) in gelatin. Some additional simplex PCR tests (targeting 126 bp bovine and 212 bp porcine mtDNA) and real-time PCR using a commercially available kit (for identification of porcine DNA) were used to verify the selectivity and sensitivity of our duplex PCR. After optimization of DNA extraction and PCR methods, hard/soft pharmaceutical gelatin capsules (containing drug) were tested for the presence of porcine and/or bovine DNA.ResultsDuplex PCR detected the presence of as little as 0.1% porcine DNA, which was more accurate than the commercially available kit. Of all gelatin capsules tested (n = 24), 50% contained porcine DNA (pure porcine gelatin alone or in combination with bovine gelatin).ConclusionsDuplex PCR presents an easy-to-follow, quick, low-cost and reliable method to simultaneously detect porcine and bovine DNAs (>100 bp) in minute amounts in highly processed gelatin-containing pharmaceutical products (with a 0.1% sensitivity for porcine DNA) which may be used for halal authentication.Graphical abstractSimultaneous detection of porcine and bovine DNA in gelatin capsules by duplex PCR

Identification Human and Animal Blood Mixtures Using Human Cytochrome b Gene

“…The identification of the sample mixtures using the cytochrome b gene could be performed until a 0.1% level [3,12].…”

Rapid Pcr–based Detection Optimization of Porcine Dna in Gelatin Capsule Shell

“…The optimization of DNA extraction from the capsule shell was summarized in table 2. The extraction was optimized from the method as described by (Nikzad et al 2017) [13] but with several replication and modification in order to obtain DNA band profile which able to be visualized in each extraction result and DNA concentration measurement. From four modifications, we were able to obtain optimum DNA amount from extraction result which gave the most acceptable result using modifications in E5, i.e.…”

“…polymerase chain reaction (PCR) method [1]. Several PCR-based methods such as PCRsouthern hybridization [1,10], polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) [11,12], real-time PCR [13], nested-PCR [14], and multiplex PCR [13] have widely established and proved that PCR-based analysis is sensitive and specific in identifying several species in low amount of DNA.…”

“…PCR-RFLP and duplex PCR, for porcine identification in gelatin capsule shells. The traces amount of DNA contained in the gelatin of the capsule shell is very low [13]. Therefore, optimization of DNA extraction methods to obtain an adequate amount of DNA for the template in PCR is required.…”

Identification of Human DNA in Mixture of Human and Chicken Blood Using PCR with Specific Primer of Cytochrome B Gene

“…Whereas, another study conducted by Shabani et al (2015) was able to detect 0.1% of pork gelatin from mixed gelatin of beef and pork. Gelatin-based capsules mixed with 0.1% pork gelatin can also be detected by Nikzad et al (2017). The thin ribbon is due to human blood (mammals) in which DNA only comes from white blood cells, whereas in chickens (non mammals) all blood cells have DNA (Alberts 2015).…”