Background: Foot and Mouth Disease (FMD) poses a significant economic threat to the cattle population in Assam, with recurrent outbreaks occurring annually. 
 
Methods: This study aimed to compare different diagnostic techniques for the molecular detection and serotyping of Foot and Mouth Disease Virus (FMDV) outbreaks in the region. Epithelial tissue samples (n=29) were collected following standard procedures. SYBR Green real-time PCR targeting the 3D gene, sandwich enzyme-linked immunosorbent assay (S-ELISA), multiplex PCR (mPCR), and reverse transcription loop-mediated isothermal amplification (RT-LAMP) were utilized for detection and serotyping. 
 
Results: All 29 tissue samples tested positive for FMDV using SYBR Green real-time PCR, mPCR, and RT-LAMP, with 100% congruence in serotyping (20 serotype O, 9 serotype A). In contrast, S-ELISA detected only 86.21% of samples as positive (17 serotype O, 8 serotype A). Statistical analysis revealed significant differences between the sensitivity of S-ELISA and molecular techniques (p = 2.91&times;10-7).The findings underscore the superior sensitivity of SYBR Green real-time PCR, mPCR, and RT-LAMP, highlighting their potential for enhancing FMDV surveillance and control efforts in Assam.

Comparing Typing Methods for Foot-and-Mouth Disease Virus Serotypes: Sandwich ELISA, Multiplex PCR, RT-LAMP and SYBR Green Real-time PCR

<div>Background: A novel, rapid and specific multiplex polymerase chain reaction was developed to diagnose hemo-parasitic infection in bovine blood co-infected with three of the most common hemo-parasites.&nbsp; 
 
Methods: The diagnostic process relied on the detection of the three different bovine hemoparasites isolated from red blood cells (RBCs) of cattle (N=30) by conventional Giemsa stained blood smear (GSBS) and confirmed by multiplex PCR. The multiplex PCR system was used to diagnose GSBS positive blood samples (N=12) found infected or co-infected with hemoparasites. The designed multiplex primer sets was attempted to amplify 205, 313 and 422 bp fragments of apocytochrome b, sporozoite and macroschizont 2 (spm2) and 16S rRNA gene for Babesia bigemina, Theileria annulata and Anaplasma marginale, respectively. 
 
Result: This multiplex PCR was sensitive with the ability to detect the presence of 150 ng of genomic DNA. The primers used in this multiplex PCR also showed highly specific amplification of specific gene fragments of each respective parasite. Comparing the two detection methods revealed that 58.33% of specimens showed concordant diagnoses with both techniques. The specificity, positive predictive value and kappa coefficient of the agreement was highest for diagnosis of B. bigemina and lowest for A. marginale. The overall Kappa coefficient for diagnosis based on GSBS for multiple pathogens compared to multiplex PCR was 0.56, slightly behind the threshold of 0.6 of agreement. Therefore, confirmation should always be based on PCR to rule out false positives due to differences in subjective observations, stain particles and false negatives due to low parasitemia. The simplicity and rapidity of this specific multiplex PCR method make it suitable for large-scale epidemiological studies and follow-up of drug treatments.</div>

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Development of Novel Multiplex PCR for Diagnosis of Co-infected Hemo-parasites in Cattle

<div>Background: In this study, we aimed to establish a multiplex fluorescence quantitative polymerase chain reaction (PCR) method for the identification and detection of bovine viral diarrhea virus (BVDV), bovine rotavirus (BRV) and bovine coronavirus (BCV). 
 
Methods: Based on the highly conserved sequences of BVDV E2 gene, BRV VP6 gene and BCV N gene in GenBank, specific primers were designed to amplify the target gene fragments of each virus and the reaction conditions and system were optimized. Multiple fluorescence quantitative methods were established by fluorescence quantitative PCR. 
 
Result: The minimum detection limits of plasmid standards for BVDV, BRV and BCV by multiplex fluorescence quantitative PCR were 1.19&times;102&nbsp;copies/&mu;L, 3.89&times;101&nbsp;copies/&mu;L and 3.74&times;101&nbsp;copies/&mu;L, respectively. The lowest sensitivity of the established method was 100 times higher than that of conventional PCR and had high sensitivity. Furthermore, BVDV, BRV and BCV were amplified specifically, with no cross-reactivity with Escherichia coli (E. coli), Salmonella and infectious bovine rhinotracheitis virus (IBRV). The intra-and inter-group coefficients of variation were less than 1%, showing good assay repeatability. Using the established method and ordinary multiplex PCR to simultaneously detect 150 clinical diarrheal disease material samples, the coincidence rate of samples with mixed infection of the three viruses was 83.3%. The results showed that the multiplex fluorescent quantitative PCR detection method established in this study provides a rapid, sensitive and specific technique for clinical diagnosis and epidemiological monitoring of BVDV, BRV and BCV.</div>

Establishment and Preliminary Application of Multiplex Fluorescent Quantitative PCR for Simultaneous Detection of BVDV, BRV and BCV

Background: The dairy sector faces formidable obstacles in the early detection of the bacteria causing subclinical mastitis and in the proactive treatment of those cases. The objective of the study was to access multiplex PCR assays (MPCR) and conventional methods for the concurrent identification of significant bacteria that cause sub-clinical mastitis and to compare these methods. 200 samples of pooled milk from cows and buffaloes in Rajasthan were gathered between 2020 and 2021.&nbsp; 
 
Methods: The primary bacterial pathogens in milk samples were identified using conventional methods such multiplex PCR, biochemical testing and culture.&nbsp; 
 
Result: From 200 combined samples of cow and buffalo milk, the traditional approach was able to extract 97 different strains. Staphylococcus aureus 54 (27%), Streptococcus spp. 30 (15%) and E. coli 13 (6.5%) were shown to be prevalent as single or mixed infections, respectively. Staphylococcus was the main pathogen that was discovered. concurrently, S. aureus, Streptococcus and E. coli. Direct detection of Staphylococcus 65 (32.5%), Streptococcus 37 (18.5%), E. coli and 16 (8%) by multiplex PCR was found in milk samples. The analysis revealed that because multiplex PCR assays have higher specificity and sensitivity than conventional procedures, they are more reliable. The multiplex PCR method employed in the current study was a simple and quick technique to identify the major pathogens and it has the potential to be a very helpful tool for determining the pathogens that cause environmental mastitis and evaluating the health of the herd.

Molecular Detection of Some Most Important Pathogenic Bacteria by Multiplex PCR for Diagnosis of Subclinical Mastitis in Bovine

Background: In India, canine Babesiosis has been emerged as a serious canine haemoprotozoan disease caused by protozoa namely Babesia vogeli and Babesia gibsoni which owe their sustainment and transmission to the tick vectors. Reliable, rapid and efficient molecular detection technique is necessary for choosing the effective treatment protocol and planning of control strategies against this emerging canine health problem. 
 
Methods: Babesia gibsoni and Babesia vogeli were detected by molecular means and characterized from extracted DNA of canine blood samples from 8 selected districts of Odisha, India, by an internally controlled multiplex PCR targeting the 18S rRNA gene.&nbsp; The assay was standardized and in-house validated using positive controls and random field samples (n=61) with history of tick infestation. The kappa values were analysed for diagnostic specificity and sensitivity of multiplex PCR assay for detection of naplasma. platys, B. gibsoni, Erhlichia canis, Hepatozoon canis and B. vogeli considering singleplex PCR counterparts as standard test. The PCR products found positive, were subjected to sequencing and phylogenetic analysis. The epidemiological survey (n=280) was performed using both microscopy and the standardized mPCR and risk factors were analyzed.&nbsp; 
 
Result: The mPCR assay generated amplicons of 602 bp and 489 bp corresponding to B. vogeli and B. gibsoni along with amplicon of 218 bp targeting canine &beta;-actin gene as IAC to avoid false negative result. The kappa values for diagnostic specificity and sensitivity of multiplex PCR assay in detection of B. gibsoni and B. vogeli revealed very good agreement with their singleplex PCR counterparts (n=61). The epidemiological survey (n=280) revealed 17.14% prevalence of Babesiosis with high prevalence of B. gibsoni in Khordha districts and incidence of infection were high in summer season. Presence of ticks and limited outdoor activity were found to be significantly associated with rise in incidence of Babesiosis. The Odisha isolates aligned well within the established genetic boundaries for these species, solidifying the utility of gene markers for epidemiological tracking and comparative studies.&nbsp;

Development of Multiplex PCR for Detection of Babesiosis in Dogs along with Molecular Characterization of Pathogens

<div>Background: White spot syndrome virus (WSSV), Infectious hypodermal and haematopoietic necrosis virus (IHHNV) and Enterocytozoon hepatopenaei (EHP) are widely prevalent diseases affecting cultured and wild shrimp. These diseases lead to significant production and economic losses. There have been reports of co-infection of WSSV with IHHNV, WSSV with EHP and EHP with IHHNV. Since there are no proper management and treatment methods, early and timely detection of diseases is crucial. This study aimed to develop a multiplex PCR assay for timely and accurate diagnosis of WSSV, IHHNV and EHP, which is essential for minimizing production and economic losses in the shrimp aquaculture industry. 
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Methods: This study involves the screening and confirmation of penaeid shrimp samples for diseases viz, WSSV, IHHNV and EHP, designing of PCR primers, optimization of PCR parameters such as annealing temperature, primer concentration and validation of the assay. 
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Result: The multiplex PCR assay detected WSSV, IHHNV and EHP in a single PCR reaction. Analytical sensitivity was found to be 100 pg/&micro;l of WSSV, 100 pg/&micro;l of IHHNV and 10 pg/&micro;l of EHP. Diagnostic sensitivity of the developed assay was 100%. Multiplex PCR assay developed in this study can be used for the screening of broodstock, post larvae, live food and environmental samples for early detection of WSSV, IHHNV and EHP.</div>

A Multiplex PCR Approach for Accurate Detection of Key Pathogens to Improve Disease Surveillance in Shrimp Aquaculture

Background: Uterine infections are a leading cause of infertility and economic loss in dairy cattle, often resulting from complex polymicrobial infections. Rapid and accurate detection of the causative pathogens is essential for effective management. This study focuses on assessing the prevalence of uterine infections and developing a multiplex PCR assay for simultaneous detection of key bacterial pathogens in cattle. 
 
Methods: A total of 1,905 cattle from the Vindhyan region were screened for uterine discharge and categorized as clear with tiny flakes, cloudy, mucopurulent, purulent, or haemorrhagic. Sixty uterine samples were aseptically collected for molecular analysis. Conventional PCR was employed to detect Escherichia coli, Fusobacterium necrophorum, Trueperella pyogenes, Clostridium tertium and Histophilus somni. A multiplex PCR (mPCR) assay was subsequently developed for simultaneous detection of E. coli, C. tertium and H. somni and its sensitivity, specificity and accuracy were assessed to evaluate diagnostic performance. 
 
Result: The overall prevalence of uterine infections was 10.44%, consisting of 60.80% subclinical endometritis, 26.63% clinical endometritis and 12.56% metritis. The prevalence rates of E. coli, C. tertium and H. somni were 85%, 92.9% and 73.33%, respectively. Conventional PCR detected E. coli in 54, F. necrophorum in 01, T. pyogenes in 70, C. tertium in 44 and H. somni in 12 of the 60 uterine samples. Using mPCR, 51 samples were positive for E. coli, 44 for C. tertium and 15 for H. somni. The sensitivity, specificity and accuracy of the mPCR were 100%, 66.6% and 95% for E. coli; 100% each for C. tertium; and 80%, 100% and 95% for H. somni, respectively, indicating its high diagnostic efficiency. 
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Prevalence of Uterine Infections and Development of a Multiplex PCR Assay for their Detection in Cattle

<div style="text-align:justify">Background: Shiga toxin-producing Escherichia coli (STEC) strains are considered to be most common food-borne enteric zoonotic pathogen, causing various disease conditions in both animals and humans and are highly pathogenic to human in low infectious doses. Resistance against antibiotics by STEC is also a big concern now a days. Hence in view of the public health significance of STEC, present work was planned to know the combination of phenotypic and genotypic resistance patterns against certain most commonly used antibiotics by using disk diffusion method and multiplex PCR respectively. 
 
Methods: A total of 426 PCR confirmed STEC isolates isolated from pooled samples (animal faecal(179), farm water(122) and human faecal samples (125) of different livestock farms in and around Proddatur andhra Pradesh, were subjected to antibiotic sensitivity test by using disc diffusion method. The isolates that showed resistance for tetracycline, streptomycin, sulphonamides and ampicillin were selected and subjected to multiplex PCR for the detection of resistance genes. 
 
Result: Disk diffusion assay revealed highest phenotypic resistance for STEC isolates against Cepahlothin (100%), followed by Ampicillin (99.06%), Tetracycline (97.2%), Streptomycin (94.3%), Sulphonamides (90.8%) and Trimethoprim (84.5%). Pooled samples also revealed the presence of antimicrobial resistance genes like tetA(59.2%), tetB(43.5%), tetC (9.2%), strA (39.3%), strB(54.1%), sul1 (40.8%), sul2 (58.7%), sul3 (3.8%) and blaTEM(83.4%). These findings indicate the highest prevalence of antimicrobial resistance among the STEC isolates, which alarms indiscriminate use of antibiotics both for therapeutic purpose and as growth promoters. Strict hygienic, sanitation and HACCP programmes should be applied to counter STEC prevalence.</div>

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Detection of Antimicrobial Resistance Genes from Shiga Toxin Producing Escherichia coli by Multiplex PCR

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Background: Tilapia Lake Virus (TiLV) is an emerging threat to global aquaculture and current PCR assays that target a single genome segment is vulnerable to false negatives due to genetic variability and viral reassortment. A robust diagnostic tool is needed for reliable detection and surveillance. 
 
Methods: We developed a multiplex polymerase chain reaction (mPCR) assay targeting three conserved genome segments (2, 3 and 8) using TiLV Primer sets that were optimized through uniplex and multiplex reactions and assay conditions were standardized at 58&deg;C. Sensitivity was assessed using serial dilutions of TiLV-positive cDNA, while specificity was evaluated against non-TiLV aquatic pathogens. Diagnostic performance was compared with semi-nested reverse transcription polymerase chain reaction (semi-nested RT-PCR) and validated using Receiver Operating Characteristic (ROC) analysis. 
 
Result: The optimized mPCR assay consistently amplified all three segments in a single reaction, producing clear and specific bands without non-specific amplification. The detection limit was 100 (picogram) pg/&micro;L of TiLV cDNA and no cross-reactivity was observed with non-TiLV pathogens. ROC analysis yielded an AUC value of 1.0, indicating perfect sensitivity and specificity. This multi-segment approach minimizes false negatives and offers a reliable tool for TiLV detection in tilapia aquaculture.

Multiplex PCR Targeting Multiple Genome Segments for Enhanced Detection of Tilapia Lake Virus (TiLV) in Tilapia

<div style="text-align: justify;">Chicken Mx protein confers resistance against viral infections including avian influenza virus. Mx protein is one of the strong antiviral proteins induced by interferon system. Different alleles of chicken Mx gene have been shown to vary in their antiviral activity against influenza virus. A non-synonymous mutation at position 2032 of chicken Mx gene responsible for variation of amino acid (Ser to Asn) in Mx protein is responsible for negative to positive antiviral activity of Mx protein. In the present study it was analyzed &nbsp;that SNP at G2032A in chicken Mx gene in two Indian native chicken breeds, Aseel, Kadaknath and compared with White Leghorn layer. A total of 90 samples, including 50 of White Leghorn and 20 each from native chicken breed were screened for SNP at position 2032 of Mx gene by using real time multiplex allele specific PCR. The resistant allele (A) frequency is higher than susceptible allele in all three chicken breeds investigated. Among the breeds, the resistant allele frequency (A) is higher in White Leghorn (0.95) followed by Aseel (0.75) and Kadaknath (0.625). Homozygous to resistant allele (AA) is the major genotype in White Leghorn whereas heterozygous to both alleles is the major genotype in Kadaknath. Variations in the gene frequency might have emerged from varying degree of selection pressure the different breeds had undergone. In future, the resistant genotype could be selected in modern breeding programs to improve the genetic status for disease resistance against viral infections.&nbsp;</div>