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Design and Optimization of a CD4 Primer for Quantitative Real-Time PCR (qRT-PCR) Puji Oktavia; Andani Eka Putra; Gestina Aliska; Zelly Dia Rofinda; Dwitya Elvira; Dessy Arisanty
Jurnal Biologi Tropis Vol. 26 No. 3 (2026): Juli - September
Publisher : Biology Education Study Program, Faculty of Teacher Training and Education, University of Mataram, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.29303/jbt.v26i3.12832

Abstract

Quantitative real-time polymerase chain reaction (qRT-PCR) is being explored as an affordable alternative to flow cytometry for monitoring CD4+ T lymphocytes in HIV-infected patients, but detailed design and validation of CD4-specific primers are rarely reported. To design a CD4-specific primer-probe set for qRT-PCR, assess its in silico specificity, and determine the optimal annealing temperature. A forward primer, reverse primer, and hydrolysis probe targeting CD4 transcript were designed from published CD4 gene sequences. Specificity was evaluated using NCBI Primer-BLAST against the Human mRNA RefSeq database. Optimal annealing temperature was determined using an 8-point thermal gradient (54.0-66.0°C) on a Bio-Rad CFX96 system with SensiFAST™ Probe No-ROX chemistry, comparing Cq values across the gradient. Results: Primer-BLAST confirmed specificity of the forward (20nt, Tm 57.42°C, GC 50.00%) and reverse (24nt, Tm 57.69°C, GC 41.67%) primers, with low self-complementarity (3.00 and 2.00) and no 3' self-complementarity. Cq values were tightly clustered (23.25-23.76) across the gradient; with the lowest Cq (23.25) at 54.8°C. The CD4-specific primer-probe set showed confirmed specificity and robust tolerance across a wide annealing temperature range, with an optimum of 54.8°C, supporting its use as a foundational tool for CD4+ T lymphocyte monitoring where flow cytometry is inaccessible.
Co-Authors - Johan Abdiana Abdiana, Abdiana Adang Bachtiar Ade Asyari Afida Razuna Ave Afrizal Agustian, Dede Rahman Aisyah Elliyanti Akmal Djamaan Alioes , Yustini Alponsin Alponsin Amelin, Fitrisia Amirah Zatil Izzah Amrin Alkamar Anavelda, Aura Putri Andre Prima Rahmadhana Annisa Widi Rizkia Antonius, Puja Agung Arfai Arfai Arfiandi Arfiandi Ariani, Tutty Arni Amir Arni Amir arniamir, Arni Amir Asri, Ennesta Bari'ah, Puti Asma Basyir, Vaulinne Besri, Hanifa Zahra Bobby Indra Utama Budi Junio Hermawan Cimi Ilmiawati, Cimi Darfioes Basir Deddy Saputra Dede Agustian Rahman Defni, - Defrin, Defrin Delfican Delfican Delfilaura, Asyifa Delmi Sulastri Densi Selpia Sopianti Densi Selpia Sopianti Desmawati Desmawati Dessy Arisanty Desy Nofita Sari Dia Rofinda, Zelly Dia Djanas, Dovy Dwitya Elvira Edison, Ebill Fuji Efrida Efrida Eka Fithra Elfi, Eka Fithra Elizabeth Bahar Elizabeth Bahar Elmatris Sy Elsa Alfiyanti Eryati Darwin Eti Yerizel Fadhil Aptana Sadjiana Fahma, Siskalil Farashanda, Meysha Farras, Umar Al Fathiyyatul Khaira Fauzul Azhim Febrianti Ika Kurnia Feby Resicha Fenty Anggraini Ferry Lismanto Syaiful Fika Tri Anggraini Finny Fitry Yani Fiona Dewanti Firdawati Firdawati Fitri Anggraini Fitri Anggraini, Fitri Fitri Nova, Fitri Fitri, Azdiana Fitrina, Dewi Wahyu Gardenia Akhyar Gestina Aliska Gestina Aliska Hafni Bachtiar Hanif, Monica Mulnia Hanif, Rayhendra Hardisman Hari, Dewi Gulyla Hasmiwati Haviz Yuad Haviz Yuad Helmizar Hidayanti, Rahmi Husna Yetti Husna, Annisa Tamara Husni Husni Ida Parwati Ifan Aulia Candra Ilmiawati, Ilmiawati Irrahmah, Miftah Irvan Medison Jamsari Jamsari Kaharudin Kaharudin Kaisar , Kevin Florentino Kosno Suprianto Leona, Denada Florencia Linosefa Linosefa Liza Fitria M. Alfian Agustian Mahata, Liganda Endo Malinda Meinapuri Maliza, Rita Marlina . Marlina Marlina Megariani Megariani Menkher Manjas Mentari Faisal Putri Meysha Farashanda Mila Permata Sari Monica Mulnia Hanif Muhammad Rayhan Firdaus Muhammad Reza Nelmi Silvia Netti Suharti Netti Suharti, Netti Nia Ayuni Putri Nila Kasuma Novita Ariani Nur Indrawaty Lipoeto Nurhayati, Nurhayati Oky Masir Prima Indra Puji Oktavia Putri Rizki Fitriani Putri, Mentari Faisal Rahmadian, Rizki Rahmat Syawqi Rahmi Lestari Rahmi, Wahida Ramadhan, Mario Arya Ranne Balqis RAVEINAL RAVEINAL Refi Amalia Utami Rheta Elkhaira Rinang Mariko Rinang Mariko Rita Hamdani Rizanda Machmud Rosfita Rasyid Roslaili Rasyid Roza Sri Yanti Russilawati, Russilawati Rustini Rustini Salmiah Agus Salmiah Agus Saptino Miro Sary, Rizka Yulia Sef Zani Meria Sekar Asri Tresnaningtyas silviayolanda, Silvia Yolanda Sinai, Nadiva Kezia Siskalil Fahma Sosmiarti Sosmiarti Sriyanti, Roza Suharti , Netti Suzana Devi Syahrul, Muhammad Zulfadli Syamel Muhammad Tarigan, Herri Novita Taufiqulhakim, Muhammad Rafi Tresnaningtyas, Sekar Asri Tyas, Syanindhita Wikanthining Utama, Bobby Indra Utami, Refi Amalia Vaulinne Basyir Wahyu Zikra Wenny Nursa Octarina Wiwik Andriani Yefri Zulfiqar Yessi Widya Putri Yosha Putri Wahyuni Yuad, Haviz Yufri Aldi Yufri Aldy Yuliati Shafan Nur Yuliati Shafan Nur Yusri Dianne Jurnalis