Simultaneous and Rapid Detection of Salmonella typhi, Bacillus anthracis, and Yersinia pestis by Using Multiplex Polymerase Chain Reaction (PCR)

Abstract

Background: Salmonella typhi, Bacillus anthracis, and Yersinia pestis are some serious human pathogens, which their early diagnosis is of great importance. Salmonella typhi, Bacillus anthracis, and Yersinia pestis cause typhoid fever, anthrax, and plague respectively. These bacteria can be used to make biologic weapons.

Objectives: In this study, we designed a new and rapid diagnostic method based on Uniplex and Multiplex PCR method.

Materials and methods: Uniplex and multiplex Polymerase Chain Reaction (PCR) were conducted on virulent genes of hp and invA of Salmonella typhimurium, Pa and chr of Bacillus anthracis, and pla of Yersinia pestis. A genome from other bacteria was used to study the specificity of the primer and the PCR test.

Results: Standard strains used in this study showed that primers were specific. As for sensitivity, it was shown that this method can diagnose 1-10 copies of the genome, or 1-10 Colony Forming Units (CFU) for each of the bacteria. All pieces except anthrax were sequenced in PCR to validate the product. DNA fragment resulted from Bacillus anthracis was confirmed by restriction enzyme digestions.

Conclusion: The designed methods are accurate, rapid, and inexpensive to find and differentiate these bacteria from similar bacteria. They can be applied for rapid diagnosis of these agents in different specimens, and bioterrorism cases.

Keywords: Bacillus anthracis; Multiplex PCR; Salmonella typhi; Yersinia pestis.

Figure 1.. Result of Uniplex and Multiplex PCR

Lane1: Yersinia pestis by primer Yer (size 520 bp); Lane2: Bacillus anthracis by primer 125 (size 1083 bp); Lane3: Salmonella typhi by primer T (size 489 bp); Lane 4: Bacillus anthracis by primer 129 (size 164 bp); Lane 5: Molecular marker (100, 200, …..500, …1000, 1100, 1200, 1300, 1400); Lane 6: Multiplex PCR with the 4 pairs special primers, Yersinia pestis by primer Yer, Bacillus anthracis by primer 125, salmonella pestis by primer T, bacillus anthracis by primer 129; Lane 7: Negative control

Figure 2.. Result of Uniplex and Multiplex PCR with the 4 Pairs Special Primers

Lane 1: Yersinia pestis by the primer Yer (size 520 bp); Lane 2: Bacillus anthracis by the primer 125 (size 1083 bp); Lane 3: Bacillus anthracis by the primer 129 (size 164 bp); Lane 4: Salmonella typhi by the primer S12; Lane 5: Molecular marker; Lane 6: Multiplex PCR by the 4 pairs special primers: Bacillus anthracis( primer 129), Salmonella typhi(primer S12), Yersinia pestis (primer Yer), Bacillus anthracis (primer 125); Lane 7: Negative control

Figure 3.. Result of Multiplex PCR with 5 Pairs Specials Primer

Lane 1: multiplex PCR, Bacillus anthracis with primer 129 (size 164 bp), Salmonella typhi with primer S12 (size 373 bp), Salmonella typhi with primer T (size 489 bp), Yersinia pestis with primer Yer (size 520 bp), and Bacillus anthracis with primer 125 (size 1083 bp); Lane 2: ladder; Lane 3: Negative control

Figure 4.. HindIII Restriction Digestion of PCR Product

Lane1: Molecular marker; Lane 2: PCR fragment; Lane 3: Product digested by HindIII enzyme; Lane4: Product digested by HindIII enzyme