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El-Enshasy, Hesham Ali
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Agriculture, Biological Sciences & Forestry Earth & Planetary Sciences

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Detection of the Yersinia enterocolitica Bacteria Targeting the myfA and ystA Genes in Contaminated Vegetable Samples using Real-Time PCR to Develop Rapid Detection of Food Poisoning Bacteria Nurjayadi, Muktiningsih; Anggraeni, Rosita GIo; Putri, Gladys Indira; Declan, Jefferson Lynford; Juliansyah, Dandy Akbar; Fahriza, Tiara; Putri, Adinda Myra Amalia; Berkahingrum, Ayu; Rahmawati, Atikah Nur; Kartika, Irma Ratna; Kurniadewi, Fera; Sukmawati, Dalia; Rahayu, Sri; Saamia, Vira; Wiranatha, I Made; Abomoelak, Bassam; El-Enshasy, Hesham Ali
HAYATI Journal of Biosciences Vol. 32 No. 4 (2025): July 2025
Publisher : Bogor Agricultural University, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.4308/hjb.32.4.989-1002

Abstract

Yersinia enterocolitica is a pathogenic bacterium with the ability to survive and multiply in food in a low-temperature environment that can cause death in humans. In previous studies, the optimum annealing temperature of ymoA, ystA, and ail gene primers with amplicons of 185 bp, 123 bp, and 192 bp, respectively, was successfully found. This study aims to develop a pathogenic bacteria detection kit with confirmation, sensitivity, and specificity of myfA and ystA primers in detecting Yersinia enterocolitica bacteria quickly and accurately using the real-time Polymerase Chain Reaction method. The results showed that myfA and ystA primers have optimum annealing temperatures at 60°C with amplicon lengths of 181 bp and 123 bp, respectively. Primer myfA was able to amplify the target with real-time PCR at Ct 12.07±1 and Tm 81±1°C, while the ystA primer at Ct 12.38±1 and Tm 83±1°C. myfA and ystA primers were also able to distinguish target and non-target bacteria based on Ct or Tm. The designed primers successfully detected Yersinia enterocolitica bacteria with the smallest concentration of 0.000439 ng/µL equivalent to 7.024 × 102 CFU. The detection limit obtained is smaller than the contamination threshold set by the Food and Drug Administration (BPOM). Primer myfA and ystA Yersinia enterocolitica also successfully detected the target bacteria in cabbage and lettuce samples artificially. Based on these results, myfA and ystA primers successfully detected Yersinia enterocolitica in vegetable samples using real-time PCR quickly, sensitively, specifically, and accurately.
Real-time PCR-based Detection of Foodborne Pathogen Cronobacter sakazakii DNA in Infant Formula Milk with Specific Targeting on the hfq Gene Nurjayadi, Muktiningsih; Juliansyah, Dandy Akbar; Declan, Jefferson Lynford; Putri, Gladys Indira; Krisdawati, Ismaya; Rahmawati, Atikah Nur; Azzahra, Maharanianska; Maulana, Irvan; Putri, Gusti Angieta; Kurniadewi, Fera; Kartika, Irma Ratna; Saamia, Vira; Wiranatha, I Made; Abomoelak, Bassam; El-Enshasy, Hesham Ali
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.32.6.1597-1607

Abstract

Cronobacter sakazakii has been linked to cause meningitis, necrotizing enterocolitis, and sepsis in infants and newborns, with case fatality rates ranging from 40 to 80%. The most common source of infection has been identified as Cronobacter sakazakii-contaminated infant formula. With a relatively specific target hfq gene, this study aims to develop a real-time PCR method to identify Cronobacter sakazakii in infant formula milk. Real-time PCR is used as a detection method because rt- PCR has higher specificity and sensitivity compared to conventional PCR methods. The real-time PCR method also has a higher level of effectiveness and time efficiency compared to conventional PCR. Cronobacter sakazakii ATCC 29544 genomic DNA was isolated and used in a real-time PCR assay. Cronobacter sakazakii DNA was amplified using a primer targeting the hfq gene, yielding a 145 bp amplicon. The results of the real-time PCR test showed that Cronobacter sakazakii DNA with a concentration of 53 ng/µL could be amplified by the primer pairs of hfq gene with Ct values of 11 respectively then had Tm values of 81.7°C±0.5. The specificity test showed that the hfq primer pairs could differentiate between the target and some non-target bacteria. The sensitivity test showed the ability of the primer to detect the smallest concentration of 3.392 pg/µL with a Ct of 26.16. Based on the results obtained, it can be concluded that the hfq primer has the potential to be used as a fast detection method for Cronobacter sakazakii bacteria in infant formula using real-time PCR.
Co-Authors Abomoelak, Bassam Anggraeni, Rosita Gio Azzahra, Maharanianska Berkahingrum, Ayu Dalia Sukmawati Fahriza, Tiara Jefferson Lynford Declan Juliansyah, Dandy Akbar Kartika, Irma Ratna Krisdawati, Ismaya Kurniadewi, Fera Maulana, Irvan Muktiningsih Nurjayadi Putri, Adinda Myra Amalia Putri, Gladys Indira Putri, Gusti Angieta Rahmawati, Atikah Nur Saamia, Vira SRI RAHAYU Wiranatha, I Made