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Journal : HAYATI Journal of Biosciences

Successful Primer Picking and Pooling for the Design of Multiplex PCR Primers Specific to Pork, Beef, Chicken, and Rat DNA Kusumawaty, Diah; Faridah, Nurul; Fibriani, Azzania; Priyandoko, Didik; Dzikrina, Hanina; Puspitasari, Diah; Tallei, Trina Ekawati; Aryani, Any
HAYATI Journal of Biosciences Vol. 31 No. 4 (2024): July 2024
Publisher : Bogor Agricultural University, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.4308/hjb.31.4.678-686

Abstract

DNA markers and Multiplex-PCR have emerged as methods for species detection in processed meat products. The primary objective of this study is to design multiplex primer sequences for pork, rat, beef, and chicken, generating distinguishable amplicons through agarose gel electrophoresis for halal detection in processed meat products. Primer design involved utilizing mitochondrial genomic data and the NCBI-Primer BLAST site to obtain specific pork and beef primer sequences. In silico simulations, including single and multiplex-PCR, were conducted using Primer Pooler. In vitro validation encompassed Single-PCR and Multiplex-PCR annealing temperature optimization, using samples of chicken, beef, pork, and rat as well as processed meat products like meatballs, sausages, and nuggets. In vitro validation demonstrated that the halal marker gene's multiplex primer efficiently amplified the target sequence, specifically at the optimal annealing temperature of 58°C. Amplicons from beef (1,217 bp), pork (860 bp), rat (622 bp), and chicken (272 bp) primers could be distinguished on a 1.5% agarose gel. The study's results can aid in cost-effective and rapid halal testing and authentication of processed meat products, offering advantages over PCR with a single primer.
Integrated DNA Barcoding and Morphometric Characterization of Palm Weevils (Rhynchophorus spp.) in North Sulawesi Balansa, Endrile Golmen; Salaki, Christina Leta; Tarore, Dantje; Mamahit, Juliet Merry Eva; Kolondam, Beivy Jonathan; Tallei, Trina Ekawati
HAYATI Journal of Biosciences Vol. 32 No. 5 (2025): September 2025
Publisher : Bogor Agricultural University, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.4308/hjb.32.5.1260-1272

Abstract

Palm weevils (Rhynchophorus spp.) are significant pests of sago palms worldwide. Yet, the taxonomy and evolutionary lineage of these species in North Sulawesi remain unclear, likely due to geographic isolation driving genetic variation and species differentiation. This study aimed to investigate the genetic diversity and morphological differentiation of Rhynchophorus across distinct geographic regions in North Sulawesi using an integrative approach combining DNA barcoding and morphometric analysis. Morphometric traits from palm weevil specimens collected in Sangihe Island, Minahasa, and Bolaang Mongondow were measured and statistically analyzed using one-way ANOVA, MANOVA, principal component analysis (PCA), and linear discriminant analysis (LDA) to assess interpopulation morphological differences. For molecular identification, the cytochrome oxidase I (COI) gene was amplified and sequenced. Phylogenetic relationships were inferred using the maximum likelihood method, and a DNA barcode gap analysis was conducted to evaluate the separation between intra- and interspecific genetic distances. Morphometric analysis revealed significant size variations among the specimens, particularly in rostrum dimensions, with the EBBM (Bolaang Mongondow) group showing the highest values. COI-gene-based identification confirmed that all specimens were of R. vulneratus. However, phylogenetic analysis showed EBMin (Minahasa) and EBBM forming a distinct subgroup, while EBMan, EBSTS, EBTam, and EBSTU (all from Sangihe Island) clustered separately. Barcode gap analysis demonstrated a clear distinction between intra- and interspecific divergence, validating COI as a reliable marker for species delimitation. This study concludes that integrating morphometric and genetic analyses reveals geographic structuring within R. vulneratus, highlighting the effectiveness of combined methods for accurate identification and population differentiation.
Comparative Study of DNA Barcoding and Mini-Barcoding based on COI Gene for Species Identification and Phylogeny of Formicidae Family (Insecta: Hymenoptera) Kolondam, Beivy Jonathan; Tallei, Trina Ekawati; Koneri, Roni; Mamahit, Juliet Merry Eva
HAYATI Journal of Biosciences Vol. 32 No. 6 (2025): November 2025
Publisher : Bogor Agricultural University, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.4308/hjb.32.6.1626-1642

Abstract

DNA barcoding and mini-barcoding are widely used to study insect biodiversity, including ants (Hymenoptera: Formicidae). This study evaluated the effectiveness of both approaches for species identification and phylogenetic inference in ants, with particular attention to the performance of universal primers during PCR amplification. Full-length DNA barcode (658 bp) and mini-barcode (127 bp) regions of the mitochondrial cytochrome c oxidase subunit I (COI) gene were retrieved from GenBank, aligned, and analysed with maximum-likelihood phylogenetics (1,000 bootstrap replicates) under the Tamura-Nei model. The universal DNA barcoding primers (Folmer's LCO1490 and HCO2198) showed generally good alignment with ant COI sequences. However, multiple nucleotide mismatches were observed in LCO1490, including one critical mismatch located within the GC clamp region. In contrast, the universal mini-barcoding primers (UniMinibarF1 and UniMinibarR1) showed critical mismatches at their 3′ ends, likely reducing PCR efficiency. Despite limitations associated with primer mismatches, both DNA barcoding and mini-barcoding are reliable for species identification. DNA barcoding and mini barcoding are both capable of distinguishing ants at the species level, regardless of the length of the sequences being compared. The same sequences have also been used for phylogenetic analysis, showing good ability to distinguish ant species based on phylogenetic tree reconstruction. The comparison of DNA barcoding and mini-barcoding in ants showed that longer sequences offered superior resolution for species identification and phylogenetic reconstruction. These findings underscore the need for primer optimisation in ant-specific applications and highlight the utility of both barcoding strategies for taxonomic and evolutionary studies.
Co-Authors Abas, Abdul Hawil Adikila, Gregorius Giani Angelina Stevany Regina Masengi Antasionasti, Irma Any Aryani Arifin, Mulyani Asep Rusyana Azzania Fibriani Balansa, Endrile Golmen Barasarathi , Jayanthi BEIVY JONATHAN KOLONDAM Celik, Ismail Daniel Febrian Sengkey Dantje Tarore Diah - Kusumawaty Diah Puspitasari Dian Handayani Diana Setya Ningsih, Diana Didik Priyandoko Dolongtelide, Jeclin Inebel Dzikrina, Hanina El-Shazly, Mohamed Elly Suoth Emran, Talha Bin Erwin Wantasen Estevam, Ethiene Castellucci Faisal, Farassa Rani Fatimawali . Florencia N. Sompie Ghazi Mauer Idroes Halimatushadyah, Ernie Hariyanto, Yuanita Amalia Herny E.I. Simbala Hizir Sofyan Idroes, Ghifari Maulana Illah Sailah Irvanizam, Irvanizam Jein Rinny Leke, Jein Rinny Kalalo, Marko Jeremia Kemala, Pati Kepel, Billy Johnson Khairan Khairan Laksono Trisnantoro Lala, Andi Lydia E. N. Tendean, Lydia E. N. Mamahit, Juliet Merry Eva Martha Marie Kaseke Masengi, Kyoko Itsuko Etsuko Gabriela Maulana, Aga Maulydia, Nur Balqis Mirda, Erisna Moh. Yani Mohd Fauzi, Fazlin Monoarfa, Alexander James Muliadi Ramli Musdalifah, Annisa Nabila, Fiki Farah Niode, Nurdjannah Jane Nurul Faridah, Nurul Patwekar, Mohsina Paulina yamlean Pendong, Christa Hana Angle Purukan, Christy Purwanto, Diana Shintawati Rahman, Sunarti Abd Ratte, Titah Amelia Rinaldi Idroes Rizkia, Tatsa Roni Koneri Runtunuwu, Stefanus Vicky Bernhard Elisa Salaki, Christina Leta Salaswati, Salaswati Sambul, Alwin Melkie Sari, Nadia Warda Sekar Sasmita, Novi Reandy Siampa, Jainer Pasca Sri Sudewi, Sri Takawaian, Agrita Feisilia Tamala, Yulianida Tania, Adinda Dwi Tendean, Lydia Estelina Naomi Teuku Rizky Noviandy Tumilaar, Sefren Geiner Turalaki, Grace Lendawati Amelia Unsratdianto Sompie, Sherwin Reinaldo Utami, Wulandari Putri Wawo, Arsianita Ester Wijaya, Puspita Wungouw, Herlina Ineke Surjane Zuchra Helwani, Zuchra Zulkarnain Jalil